Methods for in vivo delivery of nutriceuticals and compositions useful therefor

ABSTRACT

In accordance with the present invention, there are provided compositions useful for the in vivo delivery of a biologic, wherein the biologic is associated with a polymeric shell formulated from a biocompatible material. The biologic can be associated with the polymeric shell itself, and/or the biologic, optionally suspended/dispersed in a biocompatible dispersing agent, can be encased by the polymeric shell. In another aspect, the biologic associated with polymeric shell is administered to a subject, optionally dispersed in a suitable biocompatible liquid.

RELATED APPLICATIONS

This application is a continuation-in-part of application U.S. patentapplication Ser. No. 08/200,235, filed Feb. 22, 1994, now U.S. Pat. No.5,498,421, which is a continuation-in-part of U.S. patent applicationSer. Nos. 08/023,698, filed Feb. 22, 1993, now U.S. Pat. No. 5,439,686,and 08/035,150, filed Mar. 26, 1993, now U.S. Pat. No. 5,362,478, theentire contents of each of which are hereby incorporated by referenceherein.

FIELD OF THE INVENTION

The present invention relates to in vivo delivery of biologics. In oneaspect, biologic is associated with a polymeric shell formulated from abiocompatible material. The biologic can be associated with thepolymeric shell itself, and/or the biologic, optionallysuspended/dispersed in a biocompatible dispersing agent, can be encasedby the polymeric shell. In another aspect, the biologic associated withpolymeric shell is administered to a subject, optionally dispersed in asuitable biocompatible liquid.

BACKGROUND OF THE INVENTION

Microparticles and foreign bodies present in the blood are generallycleared from the circulation by the `blood filtering organs`, namely thespleen, lungs and liver. The particulate matter contained in normalwhole blood comprises red blood cells (typically 8 microns in diameter),white blood cells (typically 6-8 microns in diameter), and platelets(typically 1-3 microns in diameter). The microcirculation in most organsand tissues allows the free passage of these blood cells. Whenmicrothrombii (blood clots) of size greater than 10-15 microns arepresent in circulation, a risk of infarction or blockage of thecapillaries results, leading to ischemia or oxygen deprivation andpossible tissue death. Injection into the circulation of particlesgreater than 10-15 microns in diameter, therefore, must be avoided. Asuspension of particles less than 7-8 microns, is however, relativelysafe and has been used for the delivery of pharmacologically activeagents in the form of liposomes and emulsions, nutritional agents, andcontrast media for imaging applications.

The size of particles and their mode of delivery determines theirbiological behavior. Strand et al. [in Microspheres-BiomedicalApplications, ed. A. Rembaum, pp 193-227, CRC Press (1988)] havedescribed the fate of particles to be dependent on their size. Particlesin the size range of a few nanometers (nm) to 100 nm enter the lymphaticcapillaries following interstitial injection, and phagocytosis may occurwithin the lymph nodes. After intravenous/intraarterial injection,particles less than about 2 microns will be rapidly cleared from theblood stream by the reticuloendothelial system (RES), also known as themononuclear phagocyte system (MPS). Particles larger than about 7microns will, after intravenous injection, be trapped in the lungcapillaries. After intraarterial injection, particles are trapped in thefirst capillary bed reached. Inhaled particles are trapped by thealveolar macrophages.

Pharmaceuticals that are water-insoluble or poorly water-soluble andsensitive to acid environments in the stomach cannot be conventionallyadministered (e.g., by intravenous injection or oral administration).The parenteral administration of such pharmaceuticals has been achievedby emulsification of oil solubilized drug with an aqueous liquid (suchas normal saline) in the presence of surfactants or emulsion stabilizersto produce stable microemulsions. These emulsions may be injectedintravenously, provided the components of the emulsion arepharmacologically inert. For example, U.S. Pat. No. 4,073,943 describesthe administration of water-insoluble pharmacologically active agentsdissolved in oils and emulsified with water in the presence ofsurfactants such as egg phosphatides, pluronics (copolymers ofpolypropylene glycol and polyethylene glycol), polyglycerol oleate, etc.PCT International Publication No. WO85/00011 describes pharmaceuticalmicrodroplets of an anaesthetic coated with a phospholipid, such asdimyristoyl phosphatidylcholine, having suitable dimensions forintradermal or intravenous injection.

Protein microspheres have been reported in the literature as carriers ofpharmacological or diagnostic agents. Microspheres of albumin have beenprepared by either heat denaturation or chemical crosslinking. Heatdenatured microspheres are produced from an emulsified mixture (e.g.,albumin, the agent to be incorporated, and a suitable oil) attemperatures between 100° C. and 150° C. The microspheres are thenwashed with a suitable solvent and stored. Leucuta et al. [InternationalJournal of Pharmaceutics Vol. 41:213-217 (1988)] describe the method ofpreparation of heat denatured microspheres.

The procedure for preparing chemically crosslinked microspheres involvestreating the emulsion with glutaraldehyde to crosslink the protein,followed by washing and storage. Lee et al. [Science Vol. 213:233-235(1981)] and U.S. Pat. No. 4,671,954 teach this method of preparation.

The above techniques for the preparation of protein microspheres ascarriers of pharmacologically active agents, although suitable for thedelivery of water-soluble agents, are incapable of entrappingwater-insoluble ones. This limitation is inherent in the technique ofpreparation which relies on crosslinking or heat denaturation of theprotein component in the aqueous phase of a water-in-oil emulsion. Anyaqueous-soluble agent dissolved in the protein-containing aqueous phasemay be entrapped within the resultant crosslinked or heat-denaturedprotein matrix, but a poorly aqueous-soluble or oil-soluble agent cannotbe incorporated into a protein matrix formed by these techniques.

Thus, the poor aqueous solubility of many biologics presents a problemfor human administration. Indeed, the delivery of pharmacologicallyactive agents that are inherently insoluble or poorly soluble in aqueousmedium can be seriously impaired if oral delivery is not effective.Accordingly, currently used formulations for the delivery ofpharmacologically active agents that are inherently insoluble or poorlysoluble in aqueous medium require the addition of agents to solubilizethe pharmacologically active agent. Frequently, however, severe allergicreactions are caused by the agents (e.g., emulsifiers) employed tosolubilize pharmacologically active agents. Thus, a common regimen ofadministration involves treatment of the patient with antihistamines andsteroids prior to injection of the pharmacologically active agent toreduce the allergic side effects of the agents used to aid in drugdelivery.

In an effort to improve the water solubility of drugs that areinherently insoluble or poorly soluble in aqueous medium, severalinvestigators have chemically modified the structure of drugs withfunctional groups that impart enhanced water-solubility. Among chemicalmodifications described in the art are the preparation. of sulfonatedderivatives [Kingston et al., U.S. Pat. No. 5,059,699 (1991)], and aminoacid esters [Mathew et al., J. Med. Chem. Vol. 35:145-151 (1992)] whichshow significant biological activity. Modifications to producewater-soluble derivatives facilitate the intravenous delivery, inaqueous medium (dissolved in an innocuous carrier such as normalsaline), of drugs that are inherently insoluble or poorly soluble. Suchmodifications, however, add to the cost of drug preparation, may induceundesired side-reactions and/or allergic reactions, and/or may decreasethe efficiency of the drug.

Among the biologics which are frequently difficult to deliver is oxygen.Indeed, the need for clinically safe and effective oxygen carrying mediafor use as red blood cell substitutes ("blood substitutes" or"artificial blood") cannot be overemphasized. Some of the potential usesof such media include (a) general transfusion uses, including bothroutine and emergency situations to replace acute blood loss, (b)support of organs in vitro prior to transplantation or in vivo duringsurgery, (c) enhancing oxygen delivery to ischemic tissues and organs invivo, (d) enhancing oxygen delivery to poorly vascularized tumors toincrease the treatment efficacy of radiation therapy or chemotherapy,(e) support of organs or animals during experimental investigations, and(f) increased oxygen transport to living cells in culture media.

Blood transfusions are used to supplement the hemodynamic system ofpatients who suffer from a variety of disorders, including diminishedblood volume, or hypovolemia (e.g. due to bleeding), a decreased numberof blood cells (e.g. due to bone marrow destruction), or impaired ordamaged blood cells (e.g. due to hemolytic anemia). Blood transfusionsserve not only to increase the intravascular volume, but also to supplyred blood cells which carry dissolved oxygen and facilitate oxygendelivery to tissues.

In the case of transfusion of patients who have experienced significantblood loss, careful matching of donor and recipient blood types oftensubjects the patient to periods of oxygen deprivation which isdetrimental. Furthermore, even when autologous, patient-donated, redblood cells are available through previous phlebotomy and storage, theoxygen-carrying capacity and safety of these autologous cells declinesas a consequence of storage. Consequently, for a period of as much as 24hours after transfusion, the patient may be subject to sub-optimaloxygen delivery. Finally, there is the ever-present danger to thepatient of viral and/or bacterial contamination in all transfusions ofwhole blood and red cells derived therefrom.

Thus, there is a recognized need for a substance that is useful foroxygen transport and delivery under normal environmental conditions thatincorporates the following features. Ideally, a substance employed foroxygen transport and delivery will be capable of carrying and deliveringoxygen to devices, organs and tissues such that normal oxygen tensionsmay be maintained in these environments. Such a substance will ideallybe safe and non-toxic, free of bacterial and/or viral contamination, andnon-antigenic and non-pyrogenic (i.e. less than 0.25 EU/ml). Inaddition, the substance employed for oxygen transport and delivery willhave viscosity, colloid and osmotic properties comparable to blood. Itis also desirable that such a substance will be retained in the vascularsystem of the patient for a long period of time, thus permittingerythropoiesis and maturation of the patient's own red blood cells.Furthermore, it is desirable that the substance employed not interferewith or hinder erythropoiesis.

Currently, a number of intravenous fluids are available for thetreatment of acute hypovolemia, including crystalloids, such as lactatedRinger's solution or normal saline, and colloidal solutions, such asnormal human serum albumin. Crystalloids and colloids temporarilycorrect the volume deficit, but do not directly supplement oxygendelivery to tissues. While blood transfusion is the preferred mode oftreatment, availability of sufficient quantities of a safe supply ofblood is a perpetual problem.

Additional biologics which are frequently inherently insoluble or poorlysoluble in aqueous medium, and which are desirable to administerdissolved in an innocuous carrier such as normal saline, while promotinga minimum of undesired side-reactions and/or allergic reactions, arediagnostic agents such as contrast agents. Contrast agents are desirablein radiological imaging because they enhance the visualization of organs(i.e., their location, size and conformation) and other cellularstructures from the surrounding medium. The soft tissues, for example,have similar cell composition (i.e., they are primarily composed ofwater) even though they may have remarkably different biologicalfunctions (e.g., liver and pancreas).

The technique of magnetic resonance imaging (MRI) or nuclear magneticresonance (NMR) imaging relies on the detection of certain atomic nucleiat an applied magnetic field strength using radio-frequency radiation.In some respects it is similar to X-ray computer tomography (CT), inthat it can provide (in some cases) cross-sectional images of organswith potentially excellent soft tissue resolution. In its current use,the images constitute a distribution map of protons in organs andtissues. However, unlike X-ray computer tomography, MRI does not useionizing radiation. MRI is, therefore, a safe non-invasive technique formedical imaging.

While the phenomenon of NMR was discovered in 1954, it is only recentlythat it has found use in medical diagnostics as a means of mappinginternal structure. The technique was first developed by Lauterbur[Nature 242:190-191 (1973)].

It is well known that nuclei with the appropriate nuclear spin align inthe direction of the applied magnetic field. The nuclear spin may bealigned in either of two ways: with or against the external magneticfield. Alignment with the field is more stable; while energy must beabsorbed to align in the less stable state (i.e. against the appliedfield). In the case of protons, these nuclei precess or resonate at afrequency of 42.6 MHz in the presence of a 1 tesla (1 tesla=10⁴ gauss)magnetic field. At this frequency, a radio-frequency (RF) pulse ofradiation will excite the nuclei and change their spin orientation to bealigned against the applied magnetic field. After the RF pulse, theexcited nuclei "relax" or return to equilibrium or alignment with themagnetic field. The decay of the relaxation signal can be describedusing two relaxation terms. T₁, the spin-lattice relaxation time orlongitudinal relaxation time, is the time required by the nuclei toreturn to equilibrium along the direction of the externally appliedmagnetic field. The second, T₂, or spin-spin relaxation time, isassociated with the dephasing of the initially coherent precession ofindividual proton spins. The relaxation times for various fluids, organsand tissues in different species of mammals is well documented.

One advantage of MRI is that different scanning planes and slicethicknesses can be selected without loss of resolution. This permitshigh quality transverse, coronal and sagittal images to be obtaineddirectly. The absence of any mechanical moving parts in the MRIequipment promotes a high degree of reliability. It is generallybelieved that MRI has greater potential than X-ray computer tomography(CT) for the selective examination of tissues. In CT, the X-rayattenuation coefficients alone determine the image contrast, whereas atleast three separate variables (T₁, T₂, and nuclear spin density)contribute to the magnetic resonance image.

Due to subtle physio-chemical differences among organs and tissue, MRImay be capable of differentiating tissue types and in detecting diseasesthat may not be detected by X-ray or CT. In comparison, CT and X-ray areonly sensitive to differences in electron densities in tissues andorgans. The images obtainable by MRI techniques can also enable aphysician to detect structures smaller than those detectable by CT, dueto its better spatial resolution. Additionally, any imaging scan planecan be readily obtained using MRI techniques, including transverse,coronal and sagittal.

Currently, MRI is widely used to aid in the diagnosis of many medicaldisorders. Examples include joint injuries, bone marrow disorders, softtissue tumors, mediastinal invasion, lymphadenopathy, cavernoushemangioma, hemochromatosis, cirrhosis, renal cell carcinoma, uterineleiomyoma, adenomyosis, endometriosis, breast carcinomas, stenosis,coronary artery disease, aortic dissection, lipomatous hypertrophy,atrial septum, constrictive pericarditis, and the like [see, forexample, Edelman & Warach, Medical Progress 328:708-716 (1993); Edelman& Warach, New England J. of Medicine 328:785-791 (1993)].

Routinely employed magnetic resonance images are presently based onproton signals arising from the water molecules within cells.Consequently, it is often difficult to decipher the images anddistinguish individual organs and cellular structures. There are twopotential means to better differentiate proton signals. The firstinvolves using a contrast agent that alters the T₁ or T₂ of the watermolecules in one region compared to another. For example, gadoliniumdiethylenetriaminepentaacetic acid (Gd-DTPA) shortens the proton T₁relaxation time of water molecules in near proximity thereto, therebyenhancing the obtained images.

Paramagnetic cations such as, for example, Gd, Mn, and Fe are excellentMRI contrast agents, as suggested above. Their ability to shorten theproton T₁ relaxation time of the surrounding water enables enhanced MRIimages to be obtained which otherwise would be unreadable.

The second route to differentiate individual organs and cellularstructures is to introduce another nucleus for imaging (i.e., an imagingagent). Using this second approach, imaging can only occur where thecontrast agent has been delivered. An advantage of this method is thefact that imaging is achieved free from interference from thesurrounding water. Suitable contrast agents must be bio-compatible (i.e.non-toxic, chemically stable, not reactive with tissues) and of limitedlifetime before elimination from the body.

Although, hydrogen has typically been selected as the basis for MRIscanning (because of its abundance in the body), this can result inpoorly imaged areas due to lack of contrast. Thus the use of otheractive MRI nuclei (such as fluorine) can, therefore, be advantageous.The use of certain perfluorocarbons in various diagnostic imagingtechnologies such as ultrasound, magnetic resonance, radiography andcomputer tomography has been described in an article by Mattery [seeSPIE, 626, XIV/PACS IV, 18-23 (1986)]. The use of fluorine isadvantageous since fluorine is not naturally found within the body.

Prior art suggestions of fluorine-containing compounds useful formagnetic resonance imaging for medical diagnostic purposes are limitedto a select group of fluorine-containing molecules that are watersoluble or can form emulsions. Accordingly, prior art use offluorocarbon emulsions of aqueous soluble fluorocarbons suffers fromnumerous drawbacks, for example, 1) the use of unstable emulsions, 2)the lack of organ specificity and targeting, 3) the potential forinducing allergic reactions due to the use of emulsifiers andsurfactants (e.g., egg phophatides and egg yolk lecithin), 4) limiteddelivery capabilities, and 5) water soluble fluorocarbons are quicklydiluted in blood after intravenous injection.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are providedcompositions useful for in vivo delivery of biologics, in the form ofmicroparticles that are suitable for parenteral administration inaqueous suspension. Invention compositions comprise biologic (as asolid, liquid or gas) associated with a polymeric shell. The polymericshell is a biocompatible material, crosslinked by the presence ofdisulfide bonds. The polymeric shell associated with biologic isoptionally suspended in a biocompatible medium for administration. Useof invention compositions for the delivery of biologics obviates thenecessity for administration of biologics in an emulsion containing, forexample, ethanol and polyethoxylated castor oil, diluted in normalsaline (see, for example, Norton et al., in Abstracts of the 2ndNational Cancer Institute Workshop on Taxol & Taxus, Sep. 23-24, 1992).A disadvantage of such known compositions is their propensity to produceallergic side effects.

In accordance with another aspect of the present invention, it hassurprisingly and unexpectedly been discovered that insoluble constructsof the protein hemoglobin (Hb) prepared in accordance with the inventionreversibly bind oxygen. Insoluble hemoglobin constructs (IHC) of thepresent invention bind oxygen with oxygen affinities similar to thoseobtained with soluble hemoglobin molecules in red blood cells, orsoluble modified hemoglobin molecules that have been described in theprior art as potential blood substitutes.

In accordance with yet another aspect of the present invention, thereare provided methods for entrapping biologics in a polymeric shell.Still further in accordance with the present invention, there areprovided means for obtaining local oxygen and temperature data, and forobtaining fluorine magnetic resonance images of body organs and tissues.

The delivery of biologics in the form of a microparticulate suspensionallows some degree of targeting to organs such as the liver, lungs,spleen, lymphatic circulation, and the like, through the use ofparticles of varying size, and through administration by differentroutes. The invention method of delivery further allows theadministration of biologics, such as substantially water insolublepharmacologically active agents, employing a much smaller volume ofliquid and requiring greatly reduced administration time relative toadministration volumes and times required by prior art delivery systems(e.g., intravenous infusion of approximately one to two liters of fluidover a 24 hour period are required to deliver a typical human dose of200-400 mg of taxol).

For example, a suspension of polymeric shells of the invention can beadministered intravenously, making imaging of vascularized organs (e.g.,liver, spleen, lymph and lung) and bone marrow possible. Organ targetspecificity is achieved as a result of uptake of the micron-sizedorganofluorine-containing polymeric shells by the reticuloendothelialsystem (RES) (also known as the mononuclear phagocyte (MNP) system).Organs such as the liver and spleen play an important role in removingforeign species (e.g., particulate matter) from the bloodstream, andhence are often referred to as the "blood filtering organs". Theseorgans make up a major part of the RES. In addition, lymph nodes withinthe lymphatic circulation contain cells of the RES. Consequently,imaging of the lymphatic system is possible employing micron-sizedorganofluorine-containing polymeric shells of the present invention.Given orally or as a suppository, imaging of the stomach andgastrointestinal tract can be carried out. Such suspensions can also beinjected into non-vascular space, such as the cerebro-spinal cavity,allowing imaging of such space as well.

As a further embodiment of the present invention, paramagnetic cationssuch as Gd, Mn, Fe, and the like can be bound to polyanions, such asalginate, and used as an effective MRI contrast agent.

The present invention overcomes the drawbacks of the prior art byproviding 1) injectable suspensions of polymeric shells containingbiologic, 2) biologics in a form having enhanced stability compared tosimple emulsions, 3) organ targeting specificity (e.g., liver, spleen,lung, and the like) due to uptake of the polymeric shells of theinvention by the RES or MNP system, 4) emulsifier-free system, therebyavoiding agents that may potentially cause allergic reactions, and 5)the ability to inject relatively small doses of biologic and stillachieve good response because the biologic-containing polymeric shellsof the invention can be targeted to a specific organ.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic of a polymeric shell prepared in accordancewith the present invention. In the Figure, A refers to the insolubledisulfide crosslinked polymeric shell, B refers to the interior of thepolymeric shell, which can contain oxygen or other gas, a fluorocarboncontaining dissolved oxygen, a biocompatible oil having biologicdissolved therein, a water-in-oil emulsion containing biologic dissolvedin aqueous media, a suspension of solid particles dispersed in a liquid,and the like, C designates the thickness of the polymeric shell,typically about 5-50 nanometers, and D refers to the diameter of thepolymeric shell, typically in the range of about 0.1 up to 20 μm.

FIG. 2 presents oxygen binding curves (i.e., a graph of Hill coefficient(n) as a function of oxygen partial pressure) for a solution ofstroma-free hemoglobin (the dashed line curve) and a solution containinginsolubilized hemoglobin constructs of the present invention (the solidline curve). Actual data points with the insolubilized hemoglobinconstructs of the present invention are shown as solid boxes.

FIG. 3 presents oxygen binding curves for a solution of stroma-freehemoglobin (the dashed line curve) and a solution containinginsolubilized hemoglobin constructs of the present invention (the solidline curve) following treatment with 1.7 mM of the allosteric effector,2,3-bisphosphoglycerate (2,3-BPG). Actual data points with theinsolubilized hemoglobin constructs of the present invention are shownas solid boxes.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are providedcompositions for in vivo delivery of a biologic,

wherein said biologic is selected from:

a solid, optionally dispersed in a biocompatible dispersing agent,substantially completely contained within a polymeric shell,

a liquid, optionally dispersed in a biocompatible dispersing agent,substantially completely contained within a polymeric shell,

a gas, optionally dispersed in a biocompatible dispersing agent,substantially completely contained within a polymeric shell,

a gas associated with a polymeric shell, or mixtures of any two or morethereof,

wherein the largest cross-sectional dimension of said shell is nogreater than about 10 microns,

wherein said polymeric shell comprises a biocompatible material which issubstantially crosslinked by way of disulfide bonds, and

wherein the exterior of said polymeric shell is optionally modified by asuitable agent, wherein said agent is linked to said polymeric shellthrough an optional covalent linkage.

As used herein, the term "in vivo delivery" refers to delivery of abiologic by such routes of administration as oral, intravenous,subcutaneous, intraperitoneal, intrathecal, intramuscular, intracranial,inhalational, topical, transdermal, suppository (rectal), pessary(vaginal), and the like.

As used herein, the term "biologic" refers to pharmaceutically activeagents (such as analgesic agents, anesthetic agents, anti-asthamaticagents, antibiotics, anti-depressant agents, anti-diabetic agents,anti-fungal agents, anti-hypertensive agents, anti-inflammatory agents,anti-neoplastic agents, anxiolytic agents, enzymatically active agents,nucleic acid constructs, immunostimulating agents, immunosuppressiveagents, physiologically active gases, vaccines, and the like),diagnostic agents (such as ultrasound contrast agents, radiocontrastagents, or magnetic contrast agents), agents of nutritional value, andthe like.

As used herein, the term "micron" refers to a unit of measure of oneone-thousandth of a millimeter.

A number of biocompatible materials may be employed in the practice ofthe present invention for the formation of a polymeric shell. As usedherein, the term "biocompatible" describes a substance that does notappreciably alter or affect in any adverse way, the biological systeminto which it is introduced. Essentially any material, natural orsynthetic, bearing sulfhydryl groups or disulfide bonds within itsstructure may be utilized for the preparation of a disulfide crosslinkedshell. The sulfhydryl groups or disulfide linkages may be preexistingwithin the structure of the biocompatible material, or they may beintroduced by a suitable chemical modification. For example, naturallyoccurring biocompatible materials such as proteins, polypeptides,oligopeptides, polynucleotides, polysaccharides (e.g., starch,cellulose, dextrans, alginates, chitosan, pectin, hyaluronic acid, andthe like), lipids, and so on, are candidates for such modification.Other linkages, such as esters, amides, ethers, and the like, can alsobe formed during the ultrasonic irradiation step (so long as therequisite functional groups are present on the starting material).

As examples of suitable biocompatible materials, naturally occurring orsynthetic proteins may be employed, so long as such proteins havesufficient sulfhydryl or disulfide groups so that crosslinking (throughdisulfide bond formation, for example, as a result of oxidation duringultrasonic irradiation) can occur. Examples of suitable proteins includealbumin (which contains 35 cysteine residues), insulin (which contains 6cysteines), hemoglobin (which contains 6 cysteine residues per α₂ β₂unit), lysozyme (which contains 8 cysteine residues), immunoglobulins,α-2-macroglobulin, fibronectin, vitronectin, fibrinogen, and the like,as well as combinations of any two or more thereof.

A presently preferred protein for use in the formation of a polymericshell is albumin. Another presently preferred protein for use in theformation of a polymeric shell is hemoglobin. Yet another presentlypreferred protein for use in the formation of a polymeric shell is acombination of albumin and hemoglobin. Optionally, proteins such asα-2-macroglobulin, a known opsonin, could be used to enhance uptake ofthe shell encased particles of biologic by macrophage-like cells, or toenhance the uptake of the shell encased particles into the liver andspleen. Other functional proteins, such as antibodies or enzymes, whichcould facilitate targetting of biologic to a desired site, can also beused in the formation of the polymeric shell.

Similarly, synthetic polypeptides containing sulfhydryl or disulfidegroups are also good candidates for formation of particles having apolymeric shell. In addition, polyalkylene glycols (e.g., linear orbranched chain), polyvinyl alcohol, polyhydroxyethyl methacrylate,polyacrylic acid, polyethyloxazoline, polyacrylamide, polyvinylpyrrolidinone, and the like, are good candidates for chemicalmodification (to introduce sulfhydryl and/or disulfide linkages) andshell formation (by causing the crosslinking thereof).

In the preparation of invention compositions, one can optionally employa dispersing agent to suspend or dissolve biologic. Dispersing agentscontemplated for use in the practice of the present invention includeany liquid that is capable of suspending or dissolving biologic, butdoes not chemically react with either the polymer employed to producethe shell, or the biologic itself. Examples include water, vegetableoils (e.g., soybean oil, mineral oil, corn oil, rapeseed oil, coconutoil, olive oil, safflower oil, cotton seed oil, and the like),aliphatic, cycloaliphatic, or aromatic hydrocarbons having 4-30 carbonatoms (e.g., n-dodecane, n-decane, n-hexane, cyclohexane, toluene,benzene, and the like), aliphatic or aromatic alcohols having 1-30carbon atoms (e.g., octanol, and the like), aliphatic or aromatic estershaving 2-30 carbon atoms (e.g., ethyl caprylate (octanoate), and thelike), alkyl, aryl, or cyclic ethers having 2-30 carbon atoms (e.g.,diethyl ether, tetrahydrofuran, and the like), alkyl or aryl halideshaving 1-30 carbon atoms (and optionally more than one halogensubstituent, e.g., CH₃ Cl, CH₂ Cl₂, CH₂ Cl--CH₂ Cl, and the like),ketones having 3-30 carbon atoms (e.g., acetone, methyl ethyl ketone,and the like), polyalkylene glycols (e.g., polyethylene glycol, and thelike), or combinations of any two or more thereof.

Especially preferred combinations of dispersing agents include volatileliquids such as dichloromethane, ethyl acetate, benzene, and the like(i.e., solvents that have a high degree of solubility for thepharmacologically active agent, and are soluble in the other dispersingagent employed), along with a less volatile dispersing agent. When addedto the other dispersing agent, these volatile additives help to drivethe solubility of the pharmacologically active agent into the dispersingagent. This is desirable since this step is usually time consuming.Following dissolution, the volatile component may be removed byevaporation (optionally under vacuum).

Particles of biologic substantially completely contained within apolymeric shell, or associated therewith, prepared as described herein,are delivered neat, or optionally as a suspension in a biocompatiblemedium. This medium may be selected from water, buffered aqueous media,saline, buffered saline, optionally buffered solutions of amino acids,optionally buffered solutions of proteins, optionally buffered solutionsof sugars, optionally buffered solutions of carbohydrates, optionallybuffered solutions of vitamins, optionally buffered solutions ofsynthetic polymers, lipid-containing emulsions, and the like.

In accordance with another embodiment of the present invention, there isprovided a method for the preparation of a biologic for in vivodelivery, said method comprising subjecting medium containingbiocompatible material capable of being crosslinked by disulfide bondsand biologic to high intensity ultrasound conditions for a timesufficient to promote crosslinking of said biocompatible material bydisulfide bonds;

wherein said biologic is substantially completely contained within apolymeric shell, and

wherein the largest cross-sectional dimension of said shell is nogreater than about 10 microns.

Thus, in accordance with the present invention, biologics containedwithin polymeric shells are synthesized using high intensity ultrasound.Two non-linear acoustic processes are involved in the formation ofstable polymeric shells (i.e., acoustic emulsification and cavitation).First, acoustic emulsification disperses the biologic into the aqueousprotein solution. The dispersion formed is then chemically crosslinkedand stabilized by the formation of disulfide bonds. The disulfide bondsare formed from the cysteine residues (in the case where the polymer isa protein such as albumin) that are oxidized by superoxide which isproduced via acoustic cavitation.

The resulting suspension is optionally filtered through centriconfilters (100 kDa cutoff) and the filtered constructs or microbubbles areresuspended in normal saline or suitable buffer. FIG. 1 shows aschematic of such a construct. The average diameter of these constructsis approximately 2 microns. Particle size distribution, as determinedwith an Elzone particle counter, is seen to be quite narrow (a gaussiandistribution with a mean diameter of about 3 microns is typicallyobserved). The size range of particles obtained by this technique isbetween 0.1 micron to 20 microns. A preferred size range is 0.5 to 10microns and the most preferred range is 1 to 5 microns. This size isideally suited for medical applications, since intravenous orintraarterial injections can be accomplished without risk of small bloodvessel blockage and subsequent tissue (ischemia due to oxygendeprivation) damage. For comparison, normal red blood cells areapproximately 8 microns in diameter.

A nonobvious feature of the above-described process is in the choice ofdispersing agent, specifically with respect to the polarity of thedispersing agent. The formation of a shell about the particles ofbiologic involves reorientation of the biocompatible material at theinterface between the aqueous and non-aqueous phases such that thehydrophilic regions within the biocompatible material are exposed to theaqueous phase while the hydrophobic regions within the biocompatiblematerial are oriented towards the non-aqueous phase. In the situationwhere the biocompatible material is a protein, in order to effectunfolding, or change the conformation thereof, energy must be suppliedto the polymer. The interfacial free energy (interfacial tension)between the two liquid phases (i.e., aqueous and non-aqueous)contributes to changes in protein conformation at that interface.Thermal energy also contributes to the energy pool required forunfolding and/or change of protein conformation.

Thermal energy input is a function of such variables as the acousticpower employed in the high intensity ultrasonic irradiation process, thehigh intensity ultrasonic irradiation time, the nature of the materialbeing subjected to high intensity ultrasonic irradiation, the volume ofthe material being subjected to high intensity ultrasonic irradiation,and the like. The acoustic power of high intensity ultrasonicirradiation processes can vary widely, typically falling in the range ofabout 1 up to 1000 watts/cm² ; with an acoustic power in the range ofabout 50 up to 200 watts/cm² being a presently preferred range.Similarly, exposure time to high intensity ultrasonic irradiation canvary widely, typically falling in the range of a few seconds up to about5 minutes. Preferably, exposure time to high intensity ultrasonicirradiation will fall in the range of about 15 up to 60 seconds. Thoseof skill in the art recognize that the higher the acoustic powerapplied, the less exposure time to high intensity ultrasonic irradiationis required, and vice versa.

The interfacial free energy is directly proportional to the polaritydifference between the two liquids. Thus at a given operatingtemperature a minimum free energy at the interface between the twoliquids is essential to form the desired polymer shell. Thus, if ahomologous series of dispersing agents is taken with a gradual change inpolarity, e.g., ethyl esters of alkanoic acids, then higher homologuesare increasingly nonpolar, i.e., the interfacial tension between thesedispersing agents and water increases as the number of carbon atoms inthe ester increases. Thus it is found that, although ethyl acetate iswater-immiscible (i.e., an ester of a 2 carbon acid), at roomtemperature (˜20° C.), this dispersing agent alone will not give asignificant yield of polymer shell-coated particles. In contrast, ahigher ester such as ethyl octanoate (ester of an 8 carbon acid) givespolymer shell-coated particles in high yield. In fact, ethyl heptanoate(ester of a 7 carbon acid) gives a moderate yield while the lower esters(esters of 3, 4, 5, or 6 carbon acids) give poor yield. Thus, at a giventemperature, one could set a condition of minimum aqueous-dispersingagent interfacial tension required for formation of high yields ofpolymer shell-coated particles.

Temperature is another variable that may be manipulated to affect theyield of polymer shell-coated particles. In general the surface tensionof a liquid decreases with increasing temperature. The rate of change ofsurface tension with temperature is often different for differentliquids. Thus, for example, the interfacial tension (Δγ) between twoliquids may be Δγ₁ at temperature T₁ and Δγ₂ at temperature T₂. If Δγ₁at T₁ is close to the minimum required to form polymeric shells of thepresent invention, and if Δγ₂ (at temp. T₂) is greater than Δγ₁, then achange of temperature from T₁ to T₂ will increase the yield of polymericshells. This, in fact, is observed in the case of ethyl heptanoate,which gives a moderate yield at 20° C. but gives a high yield at 10° C.

Temperature also affects the vapor pressure of the liquids employed. Thelower the temperature, the lower the total vapor pressure. The lower thetotal vapor pressure, the more efficient is the collapse of thecavitation bubble. A more efficient collapse of the ultrasonicirradiation bubble correlates with an increased rate of superoxide (HO₂⁻) formation. Increased rate of superoxide formation leads to increasedyields of polymeric shells at lower temperatures. As a countervailingconsideration, however, the reaction rate for oxidation of sulfhydrylgroups (i.e., to form disulfide linkages) by superoxide ions increaseswith increasing temperature. Thus for a given liquid subjected toultrasonic irradiation conditions, there exists a fairly narrow range ofoptimum operating temperatures within which a high yield of polymericshells is obtained.

Thus a combination of two effects, i.e., the change in surface tensionwith temperature (which directly affects unfolding and/or conformationalchanges of the polymer) and the change in reaction yield (the reactionbeing crosslinking of the polymer via formation of disulfide linkages)with temperature dictate the overall conversion or yield of polymershell-coated particles. Temperatures suitable for the preparation ofpolymeric shells of the invention fall in the range of about 0°-80° C.

The ultrasonic irradiation process described above may be manipulated toproduce polymer shell-coated particles containing biologic having arange of sizes. Presently preferred particle radii fall in the range ofabout 0.1 up to about 5 micron. A narrow size distribution in this rangeis very suitable for intravenous delivery of biologic. The polymershell-coated particles are then preferably suspended in biocompatiblemedium (as described herein) prior to administration by suitable means.

In addition, the polymeric shell can optionally be modified by asuitable agent, wherein the agent is associated with the polymeric shellthrough an optional covalent bond. Covalent bonds contemplated for suchlinkages include ester, ether, urethane, diester, amide, secondary ortertiary amine, phosphate ester, sulfate ester, and the like bonds.Suitable agents contemplated for this optional modification of thepolymeric shell include synthetic polymers (polyalkylene glycols (e.g.,linear or branched chain polyethylene glycol), polyvinyl alcohol,polyhydroxyethyl methacrylate, polyacrylic acid, polyethyloxazoline,polyacrylamide, polyvinyl pyrrolidinone, and the like), phospholipids(such as phosphatidyl choline (PC), phosphatidyl ethanolamine (PE),phosphatidyl inositol (PI), sphingomyelin, and the like), proteins (suchas enzymes, antibodies, and the like), polysaccharides (such as starch,cellulose, dextrans, alginates, chitosan, pectin, hyaluronic acid, andthe like), chemical modifying agents (such as pyridoxal 5'-phosphate,derivatives of pyridoxal, dialdehydes, diaspirin esters, and the like),or combinations of any two or more thereof.

Variations on the general theme of dissolved biologic enclosed within apolymeric shell are possible. A suspension of fine particles of biologicin a biocompatible dispersing agent could be used (in place of abiocompatible dispersing agent containing dissolved biologic) to producea polymeric shell containing dispersing agent-suspended particles ofbiologic. In other words, the polymeric shell could contain a saturatedsolution of biologic in dispersing agent. Another variation is apolymeric shell containing a solid core of biologic produced byinitially dissolving the biologic in a volatile organic solvent (e.g.benzene), forming the polymeric shell and evaporating the volatilesolvent under vacuum, e.g., in a rotary evaporator, or freeze-drying theentire suspension. This results in a structure having a solid core ofbiologic surrounded by a polymer coat. This latter method isparticularly advantageous for delivering high doses of biologic in arelatively small volume. In some cases, the biocompatible materialforming the shell about the core could itself be a therapeutic ordiagnostic agent, e.g., in the case of insulin, which may be deliveredas part of a polymeric shell formed in the ultrasonic irradiationprocess described above. In other cases, the polymer forming the shellcould participate in the delivery of a biologic, e.g., in the case ofhemoglobin, which may be delivered as part of a polymeric shell formedin the ultrasonic irradiation process described above, thereby providinga blood substitute having a high binding capacity for oxygen.

Variations in the polymeric shell are also possible. For example, asmall amount of PEG containing sulfhydryl groups could be included withthe polymer. Upon exposure to ultrasonic irradiation, the PEG iscrosslinked into the polymer and forms a component of the polymericshell. Alternatively, PEG can be linked to the polymeric shell followingthe preparation of the shell (rather than being included as part of themedia from which the shell is prepared).

PEG is known for its nonadhesive character and has been attached toproteins and enzymes to increase their circulation time in vivo[Abuchowski et al., J. Biol. Chem. Vol. 252:3578 (1977)]. PEG has alsobeen attached to phospholipids forming the lipidic bilayer in liposomesto reduce their uptake and prolong lifetimes in vivo [Klibanov et al.,FEBS Letters Vol. 268:235 (1990)]. Thus the incorporation of PEG intothe walls of crosslinked protein shells alters their blood circulationtime. This property can be exploited to maintain higher blood levels ofbiologic and prolonged release times for the biologic.

Useful for the modification of the polymeric shell are electrophilic PEGderivatives including PEG-imidazoles, succinimidyl succinates,nitrophenyl carbonates, tresylates, and the like; nucleophilic PEGderivatives including PEG-amines, amino acid esters, hydrazides, thiois,and the like. The PEG-modified polymeric shell will be expected topersist in the circulation for longer periods than their unmodifiedcounterparts. The modification of polymeric shell with PEG may beperformed before formation of the shell, or following formation thereof.The currently preferred technique is to modify the polymeric shell afterformation thereof. Other polymers including dextran, alginates,hydroxyethyl starch, and the like, may be utilized in the modificationof the polymeric shell.

One skilled in the art will recognize that several variations arepossible within the scope and spirit of this invention. For example, thedispersing agent within the polymeric shell may be varied, a largevariety of biologics may be utilized, and a wide range of proteins aswell as other natural and synthetic polymers may be used in theformation of the walls of the polymeric shell. Applications are alsofairly wide ranging. Other than biomedical applications such as thedelivery of drugs, diagnostic agents (in imaging applications),artificial blood (sohochemically crosslinked hemoglobin) and parenteralnutritional agents, the polymeric shell structures of the invention maybe incorporated into cosmetic applications such as skin creams or haircare products, in perfumery applications, in pressure sensitive inks,pesticides, and the like.

In accordance with one embodiment of the present invention, polymericshells prepared as described above are used for the in vivo delivery ofbiologics, such as pharmaceutically active agents, diagnostic agents oragents of nutritional value (i.e. nutriceuticals). Examples ofpharmacologically active agents contemplated for use in the practice ofthe present invention include analgesic agents (e.g., acetominophen,aspirin, ibuprofen, morphine and derivatives thereof, and the like),anesthetic gases (e.g., cyclopropane, enfluorane, halothane,isofluorane, methoxyfluorane, nitrous oxide, and the like),anti-asthamatic agents (e.g., azelastine, ketotifen, traxanox, and thelike), antibiotics (e.g., neomycin, streptomycin, chloramphenicol,cephalosporin, ampicillin, penicillin, tetracycline, and the like),anti-depressant agents (e.g., nefopam, oxypertine, imipramine,trazadone, and the like), anti-diabetic agents (e.g., biguanidines,hormones, sulfonylurea derivatives, and the like), anti-fungal agents(e.g., amphotericin B, nystatin, candicidin, and the like),anti-hypertensive agents (e.g., propanolol, propafenone, oxyprenolol,nifedipine, reserpine, and the like), steroidal anti-inflammatory agents(e.g., cortisone, hydrocortisone, dexamethasone, prednisolone,prednisone, fluazacort, and the like), non-steroidal anti-inflammatoryagents (e.g., indomethacin, ibuprofen, ramifenizone, piroxicam, and thelike), anti-neoplastic agents (e.g., adriamycin, cyclophosphamide,actinomycin, bleomycin, duanorubicin, doxorubicin, epirubicin,mitomycin, methotrexate, fluorouracil, carboplatin, carmustine (BCNU),cisplatin, etoposide, interferons, phenesterine, taxol (as used herein,the term "taxol" is intended to include taxol analogs and prodrugs,taxanes, and other taxol-like drugs, e.g., Taxotere, and the like),camptothecin and derivatives thereof (which compounds have great promisefor the treatment of colon cancer), vinblastine, vincristine, as well ashormonal anti-neoplastic agents such as estrogens, progestogens,tamoxifen, and the like), anxiolytic agents (e.g., dantrolene, diazepam,and the like), enzymatically active agents (e.g., DNAse, ribozymes, andthe like), nucleic acid constructs (e.g., IGF-1 encoding sequence,Factor VIII encoding sequence, Factor IX encoding sequence, antisensenucleotide sequences, and the like), immunostimulating agents (i.e.,interleukins, interferons, vaccines, and the like), immunosuppressiveagents (e.g., cyclosporine (CsA), azathioprine, mizorobine, FK506,prednisone, and the like), physiologically active gases (e.g., air,oxygen, argon, nitrogen, carbon monoxide, carbon dioxide, helium, xenon,nitrous oxide, nitric oxide, nitrogen dioxide, and the like, as well ascombinations of any two or more thereof), as well as otherpharmacologically active agents, such as cimetidine, mitotane, visadine,halonitrosoureas, anthracyclines, ellipticine, benzocaine, barbiturates,and the like.

Examples of diagnostic agents contemplated for use in the practice ofthe present invention include ultrasound contrast agents, radiocontrastagents (e.g., iodo-octanes, halocarbons, renografin, and the like),magnetic contrast agents (e.g., fluorocarbons, lipid solubleparamagnetic compounds, GdDTPA, aqueous paramagnetic compounds, and thelike), as well as other agents (e.g., gases such as argon, nitrogen,carbon monoxide, carbon dioxide, helium, xenon, nitrous oxide, nitricoxide, nitrogen dioxide, and the like, as well as combinations of anytwo or more thereof).

Examples of agents of nutritional value contemplated for use in thepractice of the present invention include amino acids, sugars, proteins,carbohydrates, fat-soluble vitamins (e.g., vitamins A, D, E, K, and thelike) or fat, or combinations of any two or more thereof.

Key differences between the biologic-containing polymeric shell of theinvention and protein microspheres of the prior art are in the nature offormation and the final state of the protein after formation of thepolymeric shell, and its ability to carry poorly aqueous-soluble orsubstantially aqueous-insoluble agents. In accordance with the presentinvention, the polymer (e.g., a protein) is selectively chemicallycrosslinked through the formation of disulfide bonds through, forexample, the amino acid cysteine that occurs in the natural structure ofa number of proteins. An ultrasonic irradiation process is used todisperse a dispersing agent containing dissolved or suspended biologicinto an aqueous solution of a biocompatible material bearing sulfhydrylor disulfide groups (e.g., albumin) whereby a shell of crosslinkedpolymer is formed around fine droplets of non-aqueous medium. Theultrasonic irradiation process produces cavitation in the liquid thatcauses tremendous local heating and results in the formation ofsuperoxide ions that crosslink the polymer by oxidizing the sulfhydrylresidues (and/or disrupting existing disulfide bonds) to form new,crosslinking disulfide bonds.

In contrast to the invention process, the prior art method ofglutaraldehyde crosslinking is nonspecific and essentially reactive withany nucleophilic group present in the protein structure (e.g., amines,sulfhydryls and hydroxyls). Heat denaturation as taught by the prior artsignificantly and irreversibly alters protein structure. In contrast,disulfide formation contemplated by the present invention is veryspecific, and does not substantially denature the protein. In addition,particles or droplets of biologic contained within a polymeric shelldiffer from crosslinked or heat denatured protein microspheres of theprior art because the polymeric shell produced by the invention processis relatively thin compared to the diameter of the coated particle. Ithas been determined (by transmission electron microscopy) that the"shell thickness" of the polymeric coat is approximately 25 nanometersfor a coated particle having a diameter of 1 micron (1000 nanometers).In contrast, microspheres of the prior art do not have protein shells,but rather, have protein dispersed throughout the volume of themicrosphere.

The polymeric shell containing solid, liquid or gas cores of biologicallows for the delivery of high doses of biologic in relatively smallvolumes. This minimizes patient discomfort at receiving large volumes offluid and minimizes hospital stay. In addition, the walls of thepolymeric shell are generally completely degradable in vivo byproteolytic enzymes (e.g., when the polymer is a protein), resulting inno side effects from the delivery system, as is frequently the case withcurrent formulations.

According to this embodiment of the present invention, droplets orparticles of biologic are contained within a shell having across-sectional diameter of no greater than about 10 microns. Across-sectional diameter of less than 5 microns is more preferred, whilea cross-sectional diameter of about 2 microns is presently the mostpreferred for the intravenous route of administration.

In accordance with another embodiment of the present invention, thereare provided methods for preparing articles for in vivo delivery ofnutriceuticals, said method comprising subjecting aqueous mediumcontaining biocompatible material capable of being crosslinked bydisulfide bonds and a nutriceutical to high intensity ultrasoundconditions for a time sufficient to promote crosslinking of saidbiocompatible material by disulfide bonds;

wherein said nutriceutical is substantially completely contained withina polymeric shell, and

wherein the largest cross-sectional dimension of said shell is nogreater than about 10 microns.

As used herein, the term "nutriceutical" refers to an agent ofnutritional value. Examples of nutriceuticals contemplated for use inthe practice of the present invention include amino acids, sugars,proteins, Carbohydrates, fat-soluble vitamins (e.g., vitamins A, D, E,K, and the like) or fat, or combinations of any two or more thereof.

In accordance with another embodiment of the present invention, thereare provided methods for preparing articles for in vivo delivery ofnutriceuticals, said method comprising subjecting a nutriceuticalcapable of being crosslinked by disulfide bonds to high intensityultrasound conditions for a time sufficient to promote crosslinking ofsaid biocompatible material by disulfide bonds to form a polymericshell; wherein the largest cross-sectional dimension of said shell is nogreater than about 10 microns.

Articles prepared in accordance with the above methods are usefulvehicles for the administration of nutriceuticals to subjects in needthereof. As contemplated for use in the practice of the presentinvention, the articles are suitable for "in vivo delivery"administration in a variety of forms and formulations as discussedhereinbefore, including pediatric dosage formulations and formulationsfor inhalation therapy.

Children, particularly small children and infants, are an under servedpopulation when it comes to drug development. Drug dosage forms,particularly oral dosage forms such as tablets and capsules, typicallyare developed so that the mg/kg dose of drug is appropriate foradministration to adults. Frequently, it is not possible for smallchildren and infants to obtain appropriate mg/kg doses of drugs fromtablets and capsules designed for adults. For example, the dose in asingle tablet may be too large and it may not be possible to divide thetablet so that a child or infant can get the correct dose. In manycases, the child or infant cannot or will not swallow a tablet orcapsule. Many of the shortcomings inherent in tablet and capsules do notexist for liquids. Infinite dosage adjustment is possible with liquids.However, many drugs have unpleasant tastes and children and infantscannot or will not swallow them. Thus, the unpleasant tasing drugs canbe microencapsulated so that their flavor can be masked, and many drugseasily can be made into dosage forms appropriate for children andinfants. The U.S. Food and Drug Administration has recognized the needfor appropriate pediatric dosage forms and has been encouraging thepharmaceutical industry to develop them.

Therefore, we have developed a method to encapsulate mal flavored drugs,thereby masking their taste and odor. Likewise, we have been able toencapsulate pleasant flavors that are not volatized until the capsulesare physically or enzymatically degraded. Encapsulated mal flavoreddrugs can be suspended in a pleasant flavored solution that can bemetered out precisely on a mg(drug)/per kg body weight.

Particularly suitable forms of pediatric dosage formulations includesuspensions, emulsions, and chewable tablets. Suitable articlescontemplated for use in pediatric formulations contain any nutritionalagent that is suitable for administration to children. Contemplated foruse in the practice of the present invention are nutriceuticals in theform of crosslinked polymeric shells that are suitable for the sustaineddelivery of nutritional agents. As the polymeric shell degradesenzymatically or by hydrolysis, monomeric and oligomeric forms ofnutritional agents are released. As contemplated in the practice of thepresent invention, formulations that are suitable for use in inhalationtherapy include suspensions and emulsions of articles prepared by theabove methods.

In accordance with another embodiment of the present invention, it hasbeen discovered that polymeric shells as described herein, when preparedfrom hemoglobin, have surprisingly high oxygen-binding capability, andtherefore are useful as blood substitutes. Hemoglobin (Lehninger, inBiochemistry, Worth Publishers, Inc., New York, pp. 145-149, 1975) is a64,500 MW protein that consists of a tetramer (two α and two β chains).Each α and β chain binds a heme residue in a noncovalent linkage. The αand β chains are also held together by noncovalent bonds resulting fromhydrogen bonding and van der Waals forces. The four heme groups, one ineach subunit, are capable of binding four molecules of oxygen. Theseflat heme groups contain an iron atom that is in a square-planarcoordination. The four hemes are situated relatively far apart from oneanother in the intact molecule.

The interaction or cooperation of heme units in binding of oxygengreatly increases the oxygen binding capacity of each heme unit withinthe tetrameric hemoglobin molecule. In general, a single isolated hemeunit would be expected to bind a single molecule of oxygen. However,neighboring heme units within the hemoglobin molecule cooperate toincrease the bound oxygen per heme unit. This cooperativity is describedin terms of a "Hill Coefficient" whose value reflects the number ofinteracting oxygen binding sites. In the case of native hemoglobin, theHill coefficient is approximately 2.8.

Soluble hemoglobin constitutes about 90% of the total protein in redblood cells. 100 ml of whole blood is capable of absorbing approximately21 ml of gaseous oxygen due to the binding ability of hemoglobin.Equally important to the binding of oxygen, hemoglobin is also efficientin releasing the bound oxygen to tissues. The ability of hemoglobin tobind and release oxygen is often quantitatively expressed as the P₅₀ (orP_(1/2)). For example, the P₅₀ for whole blood, i.e., the partialpressure of oxygen which results in fifty percent saturation ofhemoglobin, is approximately 28 mm Hg.

The relationship between partial pressure of oxygen and percentsaturation of hemoglobin may be represented as a sigmoidal curve, theposition of which is affected by pH (the Bohr effect). The higher the pHof the hemoglobin solution at a given partial pressure of oxygen, thegreater the percent saturation with oxygen, and the lower the P₅₀ ; theoxygen saturation curve is shifted to the left on the abscissa.Conversely, the lower the pH of the hemoglobin solution, the lower thepercent saturation with oxygen, and the higher P₅₀ ; the oxygensaturation curve is shifted to the right on the abscissa. Thus, ashemoglobin moves from the relatively alkaline pH of the lungs to therelatively acidic pH of oxygen-poor tissues (producing lactic acid byanaerobic respiration), the hemoglobin molecule will have a tendency torelease its load of oxygen. Thus, in general, the affinity of hemoglobinfor oxygen changes in the opposite direction as the P₅₀ of hemoglobin.

Modifications of the hemoglobin molecule or its conformation may beassociated with changes in oxygen binding affinity. For example,association with 2,3-diphospho-glycerate (2,3-DPG, as occurs within theRBC) loosens the association between oxygen and hemoglobin, facilitatingrelease of oxygen to tissues; serum levels of 2,3 DPG rise underphysiologic conditions in which an increased delivery of oxygen isdesirable, for example, at high altitudes and during pregnancy.Oxidation of the iron ion in the heme prosthetic group from Fe(II) toFe(III) results in the formation of methemoglobin (met-Hb), which bindswater so tightly as to preclude oxygen transfer. This oxidation or`auto-oxidation` is an ongoing process in vivo which is maintained incheck by a system of redox enzymes within the red blood cell.

Hemoglobin, the protein for oxygen transport and delivery, can beseparated from the red blood cell wall membranes or stroma (stromacontain the specific antigens that determine blood type) and from othercell and plasma components. If such separation and isolation iseffected, the resulting stroma-free hemoglobin contains no antigenicmaterials; thus, blood typing and matching are no longer necessary.

Stroma-free hemoglobin (SFH), taken out of the red blood cellmicroenvironment, has been found to exhibit a propensity to bind oxygentoo tightly (a low P₅₀) and also to have a short circulating half-lifefollowing transfusion. The low P₅₀, reflective of a leftward shift inthe hemoglobin oxygen binding curve, was, in part, a consequence ofexposure of stroma-free hemoglobin to a higher pH in plasma (7.4) thanthat experienced within the erythrocyte (7.2); furthermore, the naturalassociation between hemoglobin and 2,3-diphosphoglycerate was destroyedwhen hemoglobin was removed from the red cell, thus further lowering theP₅₀. In terms of clearance from the circulation, stroma-free hemoglobinis observed to be rapidly eliminated by the kidneys, with a transfusionhalf-life (t_(1/2)) of only about 100 minutes. The Hill coefficient forSFH is in the range of 2.3-2.8.

Chemically modified hemoglobins that address some of the shortcomings ofstroma-free hemoglobin have been explored. Modifications described inthe prior art include various means for intramolecular crosslinking ofstroma-free hemoglobin; means for intermolecular crosslinking ofstroma-free hemoglobin with low molecular weight agents; means for intraand inter molecular crosslinking of stroma-free hemoglobin with lowmolecular weight agents; and means for coupling stroma-free hemoglobinto other polymers.

Methods of intramolecular crosslinking of stroma-free hemoglobin areknown in the art. See, for example, U.S. Pat. Nos. 4,584,130, 4,598,064and 4,600,531. This treatment modifies stroma-free hemoglobin bycovalently linking the lysine-99 residues on the alpha chains of theprotein through a fumarate bridge. As a consequence of thisintramolecular cross-linking, diaspirin crosslinked hemoglobin has anoxygen affinity equivalent to that of blood. Furthermore, diaspirincrosslinked hemoglobin (molecular weight 64,500) can no longer breakdown into dimers (molecular weight 32,250). As a result, the retentiontime of diaspirin alpha-alpha crosslinked hemoglobin is four to eighthours (which is two to four times that of stroma-free hemoglobin).However, this is not a sufficient length of time for utility in thetreatment of acute hemorrhage, since an oxygen carrier is needed thatcan carry oxygen for several days when the patient has lost aconsiderable amount of blood. The P₅₀ of diaspirin crosslinkedhemoglobin is in the physiological range (24-28 mm Hg) as is the Hillcoefficient (2.5-2.8).

Hemoglobin molecules have also been intermolecularly crosslinked to eachother through the use of low molecular weight crosslinking agents. Forexample, coupling of hemoglobin molecules to one another and/or to serumproteins and gelatin derivatives using dialdehydes, optimally followedby the addition of pyridoxal phosphate, is described in U.S. Pat. No.4,336,248. Crosslinking with a bifunctional or polyfunctional, lowmolecular weight crosslinking agent has been described in U.S. Pat. Nos.4,001,401, 4,001,200, 4,053,590 and 4,061,736. The products ofintermolecular hemoglobin crosslinking are often not single solubletetramers, but multiple tetramers of hemoglobin covalently linked toform soluble oligomers. Typically, products of such intermolecularcrosslinking have oxygen-carrying and delivery properties that are notequivalent to blood (P₅₀ of 18-23 for glutaraldehyde-polymerizedhemoglobin as compared to P₅₀ of 28 for whole blood) and Hillcoefficients in the range 1.8-2.8). Furthermore, prior art products ofintermolecular crosslinking by glutaraldehyde are known to be antigenic[see D. H. Marks et al., in Military Med. 152:473 (1987)].

In general, the intramolecular and intermolecular crosslinking ofhemoglobin reduces some of the renal toxicity problems that result fromthe dissociation of unmodified hemoglobin into αβ-dimers. However, thecolloidal osmotic pressure (COP) exerted by soluble hemoglobin is notsignificantly reduced by intramolecular crosslinking. This, therefore,limits the dosage level of soluble hemoglobin blood substitutes suitablefor administration. In general, an increase in COP results in a decreasein hydrostatic pressure and a concomitant decrease in the glomerularfiltration rate, resulting in oliguria and, in severe cases, anuria. Theadministration of soluble hemoglobins described in the prior art hasresulted in bradycardia, a rise in blood pressure, and a fall increatinine clearance. Vasoconstriction and tubular obstruction have beensuggested as the cause of the renal effects, which are all linked to theuse of soluble hemoglobins as blood substitutes. A highly polymerizedform of hemoglobin, such as can be prepared as described herein, whenused as a blood substitute, may alleviate these problems.

Highly fluorinated compounds, and particularly perfluorocarboncompounds, have also been considered as red blood cell substitutes, dueto their high solubilities for oxygen. Among the highly fluorinatedcompounds useful for such applications are the perfluorocarbons, e.g.,perfluorodecalin, perfluoroindane, perfluoromethyl adamantane,perfluorotripropyl amine, perfluorotributyl amine, perfluorooctylbromide, and the like. For intravenous use, these fluorocarbons, beingwater-immiscible, must be dispersed as injectible emulsions. Emulsifierstypically used in these applications are egg yolk lecithin and eggphosphatides, both of which have the potential of precipitating allergicreactions. See, for example, PCT 92/06517, which describes an emulsionthat contains a fluorochemical and phospholipids, such aslysophosphatidyl choline and lyophosphatidyl ethanolamine, assurfactants, or PCT 93/11868, which describes an emulsion with egg yolklecithin as an emulsifier that contains highly fluorinated,chloro-substituted, non cyclic organic compounds as oxygen carriers.

Fluosol-DA (Alpha Therapeutics), an emulsion of perfluorodecalin andperfluorotripropyl amine, is the only FDA approved product for use inthe prevention of transient ischemia in balloon coronary angioplasty.Another fluorocarbon product, Oxygent (Alliance Pharmaceuticals), orperfluorooctyl bromide, has approval as an oral imaging agent. Forreview of perfluoro compounds as blood substitutes, see Riess et al. inAngew Chem. Int. Ed. Engl. 17:621-634 (1978).

Blood substitutes described in the prior art contemplate only solublehemoglobins as oxygen carriers. Indeed, it is conventionally acceptedthat an insoluble hemoglobin molecule (e.g., one that is excessivelypolymerized, or crosslinked with other hemoglobin molecules to the pointof insolubility, or which is insoluble due to excessive denaturation,and the like) is not a candidate for reversible binding of oxygen, dueto the high probability of destruction or disruption of the oxygenbinding site within the molecule. In addition, the soluble hemoglobinsof the prior art have Hill coefficients which are no greater than thatof unmodified native hemoglobin.

In contrast, polymeric shells prepared from hemoglobin, as describedherein, are `giant` macroscopic molecules (due to extensivepolymerization or crosslinking of large numbers of hemoglobin tetramermolecules) which, due to the large size thereof, is insoluble in aqueousmedium. The polymerization occurs as a result of crosslinking of thesulfhydryl groups on the cysteine residues of the protein during theultrasonic irradiation process. Polymeric shell prepared in accordancewith the present invention typically comprises at least 10⁴ crosslinkedpolymer molecules, and may have as many as 10¹² hemoglobin tetramerscrosslinked into a single macroscopic `megamer` of hemoglobin. It hasunexpectedly been found that oxygen can bind reversibly to theseinsoluble constructs with affinities that are in the useful range for ared blood cell (RBC) substitute, i.e., P₅₀ between about 10 mm Hg toabout 50 mm Hg.

Another surprising and unexpected observation concerning the insolublehemoglobin construct (IHC) of the present invention is the surprisinglyhigh Hill Coefficient (n) therefor. The Hill coefficient is a measure ofthe level of cooperativity between oxygen binding sites (heme units)within the hemoglobin tetrameric molecule. The maximum Hill coefficientfor native hemoglobin is approximately 2.8, while Hill coefficientstypically reported for prior art modified hemoglobins are less than 2.8.The measured Hill coefficients for the Insoluble Hemoglobin Constructsof the present invention are extraordinarily large, typically in therange of about 5 to about 25. Without wishing to be bound by any theoryof action, these astonishingly high values can be attributed to theinteraction or communication between the oxygen binding sites of theneighboring crosslinked tetrameric hemoglobin units. Essentially, it isbelieved that the large Hill coefficient is an indication that multipletetramers cooperate in switching from the deoxy-T (tense) to the oxy-R(relaxed) state within the insoluble construct upon binding oxygen.

The unexpectedly large Hill coefficients observed in the hemoglobinconstructs of the present invention have the advantage that the amountof oxygen carried per tetramer unit of hemoglobin far exceeds thatachievable with native hemoglobin or modified hemoglobin of the priorart. This increased oxygen carrying capacity is greatly beneficial inthe utility of the invention as a RBC substitute.

The hemoglobin constructs of the present invention achieve their maximumHill coefficients at partial pressures of oxygen in the range of about40-100 mm Hg. In other words, maximum cooperativity is achieved in thisrange of oxygen pressure. Since typical alveolar pO₂ lies within thisrange, maximum uptake of oxygen from the lungs by the hemoglobinconstructs will be achieved when invention constructs are utilized as ablood substitute.

On the other hand, the release of oxygen to the tissues by the inventionconstructs is very similar to physiological hemoglobin, i.e., at typicaltissue pO₂ (<40 mm Hg), most of the oxygen bound to the insolublehemoglobin construct is released for oxygenation of the tissue. Thus,the crosslinked insoluble hemoglobin of the present invention has theunusual ability to bind oxygen at a higher capacity (due to large Hillcoefficients) than prior art hemoglobin at typical loading pressures(such as in the lungs), while retaining the ability to release oxygenefficiently at typical pressures encountered in tissue.

Due to their crosslinked nature and size, the insoluble hemoglobinconstructs of the present invention are likely to have an in vivocirculation time considerably longer than red blood cell (RBC)substitutes of the prior art. Furthermore, due to their large molecular(macroscopic) size, they are not likely to induce the renal toxicityproblems that are commonplace with conventional tetrameric or oligomericsoluble forms of hemoglobin described in the prior art.

The hollow (`bubble-like` or microbubble) insoluble hemoglobinconstructs of the present invention may be loaded with an appropriategas within the hemoglobin shell or membrane. Thus when the hemoglobin`microbubbles` are equilibriated with oxygen, e.g., in an externaldevice or within the lungs, the central core of the construct or bubbleis saturated with unbound or free oxygen that enters the core bymolecular diffusion. Thus the constructs carry unbound molecular oxygenwithin their hollow core reservoir in addition to the oxygen bound tothe hemoglobin forming the microbubble shell or membrane. The ability ofthis system to carry unbound (but entrapped) oxygen greatly increasesthe oxygen carrying capacity of the system over and above the oxygencarried by the hemoglobin alone. None of the prior art demonstrates thisability of carrying a reservoir of unbound molecular oxygen along withoxygen bound to hemoglobin.

Insoluble hemoglobin constructs can also be preloaded or saturated withoxygen prior to intravascular administration, for maximum oxygendelivery in short duration applications such as in coronary angioplastyor tumor therapy.

The discrete `cellular` nature of insoluble hemoglobin constructs of thepresent invention renders them likely to transport oxygen in aphysiologic manner, not unlike red blood cells in vivo. Due to the`megameric` nature of invention insoluble hemoglobin constructs, theywill have a colloidal osmotic pressure or oncotic pressure that isnegligible compared to an equivalent amount (in terms of oxygen carryingcapacity) of soluble hemoglobin of any of the prior art. This wouldallow for the intravenous infusion of high concentrations of inventionhemoglobin constructs, while soluble hemoglobin of the prior art may beinfused at a maximum concentration of only 6-8 g/dl for fear of severewater loss from tissues surrounding the vascular space due to osmoticgradients.

The invention lends itself to the use of other oxygen binding proteinsas RBC substitutes. As an example, the protein myoglobin, whichpossesses a single oxygen binding heme group (but no crosslinkablecysteine residues) may be expected to behave in the same way. Agenetically engineered myoglobin with at least two crosslinkablecysteine residues may be utilized to generate an insoluble myoglobinconstruct. A combination of oxygen binding proteins with proteins thathave no affinity for oxygen may be utilized in formation of theinsoluble constructs of the present invention, e.g., hemoglobin andalbumin may be used.

The invention composition has a significant advantage over encapsulatedhemoglobin compositions of the prior art. Liposomal hemoglobinformulations of the prior art comprise soluble hemoglobin within anexternal lipid layer. Liposome encapsulated hemoglobin compositions ofthe prior art suffer from several drawbacks that are overcome by theinstant invention. Leakage of soluble hemoglobin from liposomalcompositions can potentially cause nephrotoxicity. The insolubleconstructs of the present invention will not leak soluble hemoglobin dueto their extensively crosslinked nature. The aggregation of liposomes isknown to activate the complement protein C3a. This aggregation isunlikely in the case of insoluble constructs due to their size which isconsiderably larger than the liposomal size range.

The invention composition of insoluble crosslinked hemoglobin avoidstoxicity associated with soluble hemoglobin compositions of the priorart. Nephrotoxicity or renal toxicity of hemoglobin is mainly related tothe clearance of soluble dimeric, tetrameric, or oligomeric hemoglobinfrom the circulation. The hemoglobin of the instant invention, beingextensively crosslinked or `megameric`, cannot be cleared by the kidneyand is unlikely to be nephrotoxic. The insoluble constructs of theinstant invention cannot be cleared by the kidneys and thereforecircumvent this problem. An additional advantage of the extensivelycrosslinked hemoglobin constructs of the present invention over theprior art is the increased intravascular persistence due to theinsoluble form.

The morphology of the insoluble hemoglobin construct (IHC) wasdetermined using transmission electron microscopy (TEM). To obtain theTEM micrograph of a cross-sectional slice of a bovine IHC, the IHC wasfixed with glutaraldehyde, stained with osmium tetroxide and potassiumferrocyanate (to provide contrast in regions of high proteinconcentration), embedded in a low viscosity resin, and ultra-microtomed(slice thickness ˜75 nm). Since some shrinkage in the overall diameterand some shape distortion of the IHC are expected during this process,the true diameter of the IHC is best represented by the solutionparticle size distribution (3 microns; std. dev. 1), rather than directmeasurements from the TEM micrograph. A closer look at the TEMmicrograph shows three distinctive regions: a clear central region; adark, thin layer about the particle; and a loosely attached, diffuse,speckled gray region associated with the outer surface of the particle.The dark, thin layer is the IHC shell. It contains a high density ofprotein, and during staining procedure, develops the most contrast. Theloosely attached, gray matter appears to be native protein that adheresto the IHC shell during the fixation step in the sample preparation.Initial measurements from this and many other micrographs indicate theshell thickness of the bovine hemoglobin IHC to be about 25-35 nm.Hemoglobin is a roughly spherical protein (L. Stryer, Biochemistry, W.H. Freeman, New York, 1988) with a diameter of 5.5 nm. Thus, the proteinshell of the IHC is approximately 4 to 20 hemoglobin molecules(tetramers) thick. Thus, a 3.0 μm diameter bubble would contain about10⁴ to 10¹² hemoglobin molecules.

Examination of insoluble hemoglobin constructs (IHC) of the presentinvention (microbubbles or microspheres) by circular dichroism revealedthat the content of alpha-helices and beta-pleated sheets in the IHC wasnot significantly different from that of purified stroma free hemoglobin(SFH). This observation is significant because it indicates that thecrosslinking procedure and formation of insoluble hemoglobin does notresult in denaturation (i.e., the alteration of the tertiary andquaternary structure) of the protein. This observation, of course, iscorroborated by functional data showing the retention of reversibleoxygen binding and cooperativity between oxygen binding heme units afterthe synthetic step.

The oxygen binding properties of the IHC have been determined. Sincehemoglobin in the met-Fe(III) form cannot bind oxygen, the reductionsystem of Hyashi et al. (A. Hyashi, T. Suzuki, M. Shin. Biochim.Biophys. Acta 310:309, 1973) was used to reduce Fe(III) to Fe(II). Thereduction system consists of various concentrations ofglucose-6-phosphate, glucose-6-phosphate dehydrogenase, NADP,ferredoxin, ferredoxin reductase and catalase. Before each oxygenbinding experiment, the reduction system was added to the IHC andremained at 4° C. for 24-36 hours.

A bovine and human hemoglobin IHC were synthesized as described inExample 14. As recognized by those of skill in the art, the hemoglobinemployed can be derived from any vertebrate, invertebrate or eukaryoticsource, or can be the product of genetic manipulation of vertebrate,invertebrate or eukaryotic cells. Table 1 provides a summary of thecurrent results.

                  TABLE 1                                                         ______________________________________                                        Summary of n.sub.max and P.sub.50 Values of Sonicated Hb Microbubbles         and Unsonicated BHb in Various Concentrations of Phosphates                   Effector                                                                            Sonicated Hb Microbubbles                                                                       Unsonicated Hb Solution                               Concen.                                                                             IHP        2,3-BPG    IHP      2,3-BPG                                  (mM)  n.sub.max                                                                            P.sub.1/2                                                                             n.sub.max                                                                          P.sub.1/2                                                                           n.sub.max                                                                          P.sub.1/2                                                                           n.sub.max                                                                          P.sub.1/2                     ______________________________________                                        0     9.5    21.2    9.5  21.2  2.7  22.3  2.7  22.3                          0.25  12.1   22.2    11.5 22.0  2.7  24.7  2.7  22.5                          0.5   15.2   28.3    13.0 25.1  2.8  28.2  2.8  23.2                          1.0   15.1   32.1    13.4 28.7  2.8  30.2  2.8  24.9                          1.7   17.6   39.5    14.0 32.6  2.8  34.1  2.8  28.0                          ______________________________________                                         Note:                                                                         The Hill coefficients (n) for the BHb microbubbles are calculated from th     formula:                                                                      ##STR1##                                                                      where Y is the fraction oxygenated and P.sub.O.sbsb.2 is the oxygen           pressure                                                                      For the microbubbles, each Δ log (Y/1-Y) term is averaged over five     consecutive points.                                                      

All binding experiments were done at 25° C. in Tris-buffer (pH 7.4). TheIHC retain their ability to bind oxygen reversibly, as demonstrated byUV-visible spectra of the IHC, which indicates the presence ofmet-Fe(III), oxy-Fe(II) and deoxy-Fe(II) forms. The IHC can be cycledbetween the deoxy and oxy states for more than ten cycles withoutsubstantial degradation. This is important because it indicates that theenvironment surrounding the active heme site has not been alteredsignificantly in the process of making the IHC red blood cellsubstitute.

These oxygen binding data suggest that the IHC comprises substantiallynon-denatured hemoglobin. If it was denatured, no physiological (orless) reactivity would be observed.

Oxygen binding curves for the reduced hemoglobin IHC and native stromafree hemoglobin in the absence of phosphates are sigmoidal in shape,indicating cooperativity in oxygen binding. The P₅₀ values (the pressureat which half of the available binding sites on hemoglobin are bound byoxygen) are similar in both curves (21 torr versus 22 torr). This resultindicates that the IHC bind and release oxygen at similar oxygenpressures as native hemoglobin. Strikingly, the maximum Hillcoefficient, n_(max) (indicating the level of cooperativity betweenoxygen binding sites) of the IHC is significantly higher than thestroma-free hemoglobin solution (9.5 versus 2.6; see FIG. 2). Hillcoefficients (n) were calculated using the formula: ##EQU1## where:Y=fraction oxygenated, and

P_(O2) =oxygen partial pressure

Some smoothing was done by averaging each (delta) log (Y/1-Y) term overfive consecutive points.

Allosteric effectors of native hemoglobin such as inositol hexaphosphate(IHP) and 2,3-bisphosphoglycerate (2,3-BPG) have been shown to increaseboth P₅₀ (i.e. lower oxygen affinity) and to enhance cooperativity. Thesame effects are seen in the IHC. Even though the P₅₀ values areincreased by the same amount, a more dramatic effect is seen in thecooperativity of the IHC. The n_(max) increased dramatically over thatof native hemoglobin in the presence of 1.7 mM IHP (17.6 vs. 2.8) and2,3-BPG (14 vs. 2.8) (see FIG. 3 and Table 1).

This unexpectedly large increase in cooperativity apparently is due tothe covalent bonding between hemoglobin tetramers within the IHC shell.The Hill coefficient cannot be greater than the number of interactingbinding sites. The values of approximately 2.8 in native hemoglobinreflects the cooperativity in one tetramer. However, in the IHC shell,there is communication between several of the cross-linked tetramers(from the formation of disulfide bonds) upon binding oxygen. Theinteractions with nearest-neighbor tetramers are likely to be strongest;however, additional weaker interactions between tetramers further awaymay exist. Essentially, the large n_(max) is an indication that multipletetramers cooperate in switching from deoxy-T to the oxy-R state withinthe IHC shell upon binding oxygen. Again, TEM micrographs of hemoglobinIHC reveal a shell thickness of about six hemoglobin tetramers. A 3.0 μmdiameter bubble would contain about 10⁴ to 10¹² hemoglobin molecules.

Stability upon storage of the IHC was tested by particle counts atvarious time periods after preparation. The IHC were stored in sterilesaline at 4° C. for up to 6 months. At 3 months, the concentration ofthe IHC had decreased by about 10%, while at 6 months the concentrationhad dropped by about 25-30%.

The auto-oxidation rate of the IHC (from oxy-Fe(II) to met-Fe(III)) hasbeen determined to be greater than 60 hours, 96 hours, and 25 days at37° C., 25° C. and 4° C., respectively. No special precautions weretaken to maintain an inert atmosphere when these results were obtained.The prior art clearly demonstrates the benefit of maintaining an inertatmosphere such as nitrogen to decrease the rate of auto oxidation ofhemoglobin. Storage under such conditions would be expected to greatlyincrease the fraction of Fe(II) hemoglobin maintained over a longer timeperiod.

In addition, auto-oxidation may be prevented by storage of the IHCsuspension with the reduction system of Hyashi et al. described above.

Pasteurization was investigated as a method of end stage sterilizationfor the IHC suspensions. Several different pasteurization conditionswere utilized. Particle counts after each condition were used todetermine any deleterious effects of temperature on the IHC.

Condition 1: Temperature of the IHC suspension was ramped from 25°-62.8°C. in 8 minutes and held at this temperature for 30 min. Particle countsshowed a degradation of less than 20%.

Condition 2: Temperature of the IHC suspension was ramped from 25°-71.7°C. in 10 minutes and held at this temperature for 15 seconds. Particlecounts showed a degradation of less than 20%.

Condition 3: Temperature of the IHC suspension was ramped from 25°-89.5°C. in 12 minutes and held at this temperature for 2 seconds. Particlecounts showed a severe degradation of greater than 70%.

Thus, conditions 1 and 2 were found to be suitable as pasteurizationmodes. Gamma radiation as an end stage sterilization modality is alsosuitable.

The oxygen affinity (or P₅₀) of the IHC may altered by chemicalmodification of the hemoglobin with known allosteric effectors. Ingeneral, the modification of hemoglobin restricts the transition betweenthe two oxy and deoxy conformations, so the oxygenation function isalmost always altered in some way. For example if hemoglobin is modifiedin the oxy form, high oxygen affinity is usually favored, while thereverse is true if the modification is carried out in the deoxycondition. Derivatives of pyridoxal are useful modifiers since thismolecule mimics the natural allosteric effector 2,3-diphosphoglycerate(DPG). They bind to the terminal amino groups of hemoglobin. For examplethe hemoglobin may be reacted with pyridoxal 5'-phosphate (PLP) thatmimics the natural interaction of 2,3-DPG to increase the P50. Otherderivatives of pyridoxal such as 2-nor-2-formyl PLP, a bifunctionalagent that links the hemoglobin β chains, or bis-pyridoxaltetraphosphate are useful modifiers. Other crosslinkers such as acyltris(sodium methyl phosphates) may also be utilized to crosslink the βchains.

Aldehyde modifiers may also be used. For example glutaraldehyde isuseful in polymerization of hemoglobin and can be used in conjunctionwith PLP.

Diaspirin esters such as 3,5-bis (dibromosalicyl)fumarate and thecorresponding monoaspirin are useful allosteric modifiers. The aspirinbinds between the a chains of hemoglobin and the monofunctional reagentto an internal lysine. Both increase the P₅₀ of hemoglobin.

Thus `low affinity` or `high affinity` constructs may be prepared forapplication in situations other than in cases of trauma and acute bloodloss, such as in situations where local delivery of oxygen is requiredand beneficial.

A `low affinity` construct, i.e., one with a high P₅₀ (>28 mm Hg),produced by the technique above has utility in the use of oxygen as anadjuvant in the treatment of tumors by radiation or chemotherapy. Suchconstructs are loaded to maximum oxygen capacity outside the body andthen administered to the circulation of the tumor. This allows for alarge amount of oxygen release at the tumor. Activated oxygen producedin the presence of radiation or chemotherapy results in greatercytotoxic activity at the tumor site.

A `high affinity` construct (P₅₀ <28 mm Hg) has utility for `IschemicOxygen Delivery` Ischemia, or oxygen deprivation of tissue may occur ina number of pathological conditions, e.g., stroke, myocardialinfarction, and the like. The preferential release of oxygen in suchareas would help minimize permanent tissue damage. An oxygen carrier orRBC substitute with oxygen affinity similar to whole blood will notpreferentially release oxygen at such a site. However, one with a highoxygen affinity (i.e., a low P₅₀ compared to whole blood), whileretaining most of its oxygen under conditions of normally encounteredoxygen gradients, will preferentially release its oxygen at such anischemic site due to the large oxygen gradient between the blood andtissue. The affinities of Insoluble hemoglobin constructs of the presentinvention may easily be manipulated to a suitable value (P₅₀) for suchapplication by changing the nature of crosslinking, by using a suitablenatural hemoglobin with the desired affinity, or by using a geneticallyengineered hemoglobin of suitable affinity.

The insoluble hemoglobin constructs of the present invention canencapsulate and thereby act as effective carriers of pharmacologicalagents such as oxygen carriers (e.g., fluorocarbons), drugs, diagnosticagents, and the like. The encapsulated fluorocarbons (FC) are effectiveoxygen carriers that transport and release dissolved oxygen in a linearrelationship to the partial pressure of oxygen while the hemoglobinshell of the IHC transports and releases bound oxygen in a sigmoidalrelationship to oxygen pressure. This unique combination of hemoglobinand fluorocarbon within the same formulation allows for maximaltransport and release of oxygen in vivo.

The ability to deliver hemoglobin (Hb) and fluorocarbon (FC)simultaneously has not been disclosed in the prior art. Encapsulatedfluorocarbon within the core of the hemoglobin shell is capable ofacting as an oxygen reservoir. This combination allows for the deliveryof oxygen bound to the carrier in a sigmoidal relationship with pressure(i.e., for hemoglobin) as well as linear relationship to pressure (i.e.,for the fluorocarbon). This combination allows for the `background`release of oxygen in a linear fashion (from fluorocarbon) with respectto tissue pO₂ and `bolus` release of oxygen in a sigmoidal fashion (fromhemoglobin) with respect to tissue pO₂. This allows for a more efficientoxygen delivery especially in cases where large amounts of oxygen are tobe delivered for short periods, e.g., in tissue ischemia or tumortherapy.

The Hb/FC combination has the added advantage of external monitoring asto the localization of the intravascularly delivered dose. Since the ¹⁹F nucleus is easily imaged by magnetic resonance imaging (MRI), it ispossible to trace the accumulation of the delivered suspension withinthe vasculature and the tissue. This has great advantages in tumortreatment where oxygen is used as an adjuvant with radiation orchemotherapy to precisely monitor the delivery of the oxygen-carryinghemoglobin/FC suspension to the desired site.

A number of fluorocarbons (FCs) are suitable for use in the practice ofthe present invention, as described in detail below.

Furthermore, proteins that have no oxygen binding capabilities but havecrosslinkable cysteine residues or sulfhydryl groups (native orartificially introduced) may be used to encapsulate biocompatiblefluorocarbons with suitable oxygen affinities for use as bloodsubstitutes. As an example, albumin can be used to encapsulateperfluorodecalin or perfluorotripropylamine for use as a bloodsubstitute.

Several drugs are candidates for encapsulation in hemoglobinmicrospheres of the present invention. Several chemotherapeutic agentsrequire the presence of oxygen for maximal tumor cytotoxicity. Thedelivery of such drugs within constructs of an oxygen carrier such ashemoglobin effectively combines the essential components of cytotoxicityinto a single package. Several useful cytotoxic drugs are oil-soluble.These drugs may be dissolved in a fluorocarbon or other biocompatibleoil such as soybean oil, safflower oil, coconut oil, olive oil, cottonseed oil, and the like. The oil/drug solution is subjected to ultrasonicirradiation with a hemoglobin solution to produce microspheres ofoil/drug within a shell of crosslinked insoluble hemoglobin. Thesuspension may be oxygenated prior to intravascular administration.Oil-soluble cytotoxic drugs include cyclophosphamide, BCNU, melphalan,mitomycins, taxol and derivatives, taxotere and derivatives,camptothecin, adriamycin, etoposide, tamoxifen, vinblastine, vincristineand the like; nonsteroidal antiinflammatories such as ibuprofen,aspirin, piroxicam, cimetidine, and the like; steroids such as estrogen,prednisolone, cortisone, hydrocortisone, diflorasone, and the like,drugs such as phenesterine, mitotane, visadine, halonitrosoureas,anthrocyclines, ellipticine, diazepam, and the like; immunosuppressiveagents such as cyclosporine, azathioprine, FK506, and the like.

Water-soluble drugs may also be encapsulated within the IHC shell by amethod of double emulsion. First, an aqueous drug solution is emulsifiedwith a biocompatible oil to obtain a water-in-oil (W/O) emulsion. TheW/O emulsion is treated as an oil phase and subjected to ultrasonicirradiation with an aqueous hemoglobin solution as above to produce IHCcontaining within their shell, a microemulsion of the desiredwater-soluble drug. Emulsifiers contemplated for use in this embodimentof the present invention include the Pluronics (block copolymers ofpolyethylene oxide and polypropylene oxide), phospholipids of egg yolkorigin (e.g., egg phosphatides, egg yolk lecithin, and the like); fattyacid esters (e.g., glycerol mono- and di-stearate, glycerol mono- anddi- palmirate, and the like). Water-soluble drugs contemplated for usein this embodiment of the present invention include antineoplastic drugssuch as actinomycin, bleomycin, cyclophosphamide, duanorubicin,doxorubicin, epirubicin, fluorouracil, carboplatin, cisplatin,interferons, interleukins, methotrexate, mitomycins, tamoxifen,estrogens, progestogens, and the like.

The double emulsion technique is also suitable for delivery of otheraqueous-soluble material of therapeutic, diagnostic or nutritionalvalue. For example, the hemoglobin content of the IHC may be increasedby encapsulating a hemoglobin microemulsion into the IHC.

In order to make the IHC in a greater likeness to red blood cells, aphospholipid bilayer can be formed around the crosslinked hemoglobinmicrobubbles. Such a bilayer results in the formation of a true `redcell analog` and may be created in a two step process. Chargedphospholipids or lipids utilized in the formation of this bilayerinclude phosphatidyl choline, phosphatidyl ethanol amine, phosphatidylserine, phosphatidyl inositol, phosphatidyl glycerol, sphingomyelin,dimyristoylphosphatidic acid, dipalmitoyl phosphatidic acid,sarcosinates (sarcosinamides), betaines, monomeric and dimeric alkyds,and the like. Nonionic lipids may also be utilized in this invention,including polyethylene fatty acid esters, polyethylene fatty acidethers, diethanolamides, long chain acyl hexosamides, long chain acylamino acid amides, long chain amino acid amines, polyoxyethylenesorbitan esters, polyoxy glycerol mono- and di-esters, glycerol mono-and di-stearate, glycerol mono- and di-oleate, glycerol mono- anddi-palmitate, and the like.

Another variation on this technique is to utilize photopolymerizablelipids or lipids that may be readily crosslinked via a chemical reactionin order to provide a more stable lipid `membrane` coat.Photopolymerizable lipids that may be utilized in the present inventioninclude acrylate or methacrylate substituted lipids (such asphosphatidyl choline, phosphatidyl ethanol amine, phosphatidyl serine,phosphatidyl glycerol, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, and the like); lipids with native polymerizableunsaturation (such as unsaturated phosphatidyl cholines with diacetylenegroups or conjugated diene groups, and the like), and so on. Lipids thatreadily undergo crosslinking via thiol-disulfide exchange also are goodcandidates for the formation of a stable lipid coat for the IHC.Examples of such lipids include derivatives of phosphatidyl cholinesesterified with lipoic acid, and the like.

IHCs synthesized by ultrasonic irradiation can be administered as asuspension in a biocompatible medium, as described above, as well asother agents of nutritional value.

Preferred routes for in vivo administration are the intravenous,intraarterial, intramuscular, subcutaneous, intraperitoneal, oral,inhalational, topical, transdermal, suppository, pessary and the like.

In summary, the insoluble hemoglobin constructs of the present inventionhave numerous advantages over prior art soluble hemoglobin, prior artencapsulated soluble hemoglobin, and prior art fluorocarbon bloodsubstitutes or oxygen carriers. These advantages include:

higher oxygen capacity;

variable oxygen affinity;

insoluble `megameric` hemoglobin, which is expected to persist longer incirculation than prior art tetrameric or oligomeric soluble hemoglobin;

lower potential of kidney toxicity due to large molecular size;

less likely to leak hemoglobin than in the case of liposome encapsulatedhemoglobin;

due to much larger size than liposomes, formation of aggregates thatstimulate complement proteins is unlikely;

behaves more like RBC due to discrete `cellular` nature compared tosoluble hemoglobin of prior art;

can carry a reservoir of unbound oxygen along with oxygen bound tohemoglobin;

can be used as a fluorocarbon (FC) carrier without potentially allergicand toxic emulsifiers;

crosslinked hemoglobin in Hb/FC constructs provides for enhancedstability relative to prior art emulsified systems that use eggphosphatides and/or other synthetic surfactants;

release profiles of oxygen from Hb/FC is a combination of sigmoidal andlinear in relation to tissue pO₂ ;

Hb/FC constructs can be detected and monitored in vivo by ¹⁹ F MRI;

hemoglobin or Hb/FC constructs may be used as drug carriers in additionto carrying oxygen;

a lipid bilayer membrane may be applied to the hemoglobin construct tomake it appear more physiological;

the hemoglobin construct may be modified with polymers such as PEG tofurther increase intravascular persistence.

In accordance with yet another aspect of the present invention, it hasbeen found that organofluorine-containing compounds, which in generalare hydrophobic, water immiscible and consequently difficult toadminister, can be entrapped in polymeric shells (as described above)for ease of delivery. Organofluorine-containing compounds entrappedwithin polymeric shells are readily usable and biocompatible. Theparticle size of polymeric shells produced in accordance with thepresent invention have an average diameter of approximately 2 microns,which is ideal for medical applications, since intravenous orintraarterial injections can be accomplished without risk of small bloodvessel blockage and subsequent tissue damage (e.g., caused by ischemiadue to oxygen depravation). For comparison, red blood cells areapproximately 8 microns in diameter (thus injectable biomaterial shouldbe smaller than 8-10 microns in diameter to prevent blood vesselblockage).

Naturally occurring fluorine atoms (¹⁹ F) give a clear nuclear magneticresonance signal and thus can function as contrast agents or "probes" inMRI. The specific advantages for the use of ¹⁹ F include: 1) anextremely low native concentration in the body (fluorine is notnaturally found in the body), 2) a high nuclear magnetic resonancesensitivity, 3) a magnetogyric ratio close to that of ¹ H, thuspermitting ¹⁹ F magnetic resonance imaging to be carried out with onlyminor modifications of existing MRI devices, and 4) low toxicity of mostorganofluorine-containing compounds.

In general, fluorocarbons are non-toxic and biocompatible. Fluorocarbonsare stable and unreactive, and consequently are not likely to bemetabolized due to their strong carbon-fluorine bonds (approximately 130kcal/mole). For comparison, carbon-hydrogen bonds (approximately 100kcal/mole) are weaker and much more reactive. The FDA has approved twofluorocarbons, perfluorotripropyl amine and perfluorodecalin, formedicinal use as blood substitutes under the trade name of Fluosol DA.

A number of different fluorocarbons can be used in the practice of thepresent invention. For example, compounds satisfying the followinggeneric formulae can be incorporated into polymeric shells employing theinvention procedure as described herein:

(a) C_(x) F_(2x+y-z) A_(z), wherein:

x=1-30, preferably 5-15,

y=2; or 0 or -2, when x ≧2; or -4 when x ≧4,

z=any whole number from 0 up to (2x+y-1), and

A is selected from H, halogens other than F, --CN, --OR, wherein R is H,alkyl, fluoroalkyl, alkenyl, fluoroalkenyl, alkynyl, fluoroalkynyl,aryl, fluoroaryl, alkanoyl, fluoroalkanoyl, alkenoyl, fluoroalkenoyl,alkynoyl, fluoroalkynoyl,

(b) [C_(x) F_(2x+y),_(-z) A_(z) ]_(a) JR_(b-a), wherein:

x, z, A and R are as defined above,

y'=+1; or -1 or -3, when x ≧2; or -5 when x≧4,

J=O, S, N, P, A1, or Si,

a=1, 2, 3, or 4, and

b=2 for a divalent J, or 3 for a trivalent J, 4 for a tetravalent J,

(c) A'--[(CF₂)_(x) --O]_(c) -A", wherein:

x is as defined above,

A' is selected from H, halogens, --CN, --OR, wherein R is H, alkyl,fluoroalkyl, alkenyl, fluoroalkenyl, alkynyl, fluoroalkynyl, aryl,fluoroaryl, alkanoyl, fluoroalkanoyl, alkenoyl, fluoroalkenoyl,alkynoyl, fluoroalkynoyl,

A" is selected from H or R, wherein R is as defined above,

c=1-200, preferably 2-50, or ##STR2## wherein: x is as defined above,and

c'=2-20, preferably 2-8,

as well as mixtures of any two or more thereof.

Included within the above generic formulae are compounds having generalformulae such as:

C_(x) F_(2x), such as, for example, perfluoro-1-hexene (C₆ F₁₂),perfluoro-2-hexene (C₆ F₁₂), perfluoro-3-hexene (C₆ F₁₂), and the like,

cyclo-C_(x) F_(2x), such as, for example, perfluorocyclohexane (C₆ F₁₂),perfluorocyclooctane (C₆ F₁₆), and the like,

C_(x) F_(2x-2), such as, for example, perfluoro-1-hexyne (C₆ F₁₀),perfluoro-2-hexyne (C₆ F₁₀), perfluoro-3-hexyne (C₆ F₁₀), and the like,

bicyclo- C_(x) F_(2x-2), such as, for example, perfluorodecalin (C₉F₂₀), and the like,

C_(x) F_(2x+2), such as, for example, perfluorohexane (C₆ F₁₄),perfluorooctane (C₆ F₁₈), perfluorononane (C₉ F₂₀), perfluorodecane (C₁₀F₂₂), perfluorododecane (C₁₂ F₂₆), and the like,

C_(x) F_(2x-4), such as, for example, perfluoro-2,4-hexadiene, and thelike,

C_(x) F_(2x+1) A, such as, for example, perfluorotripropyl amine [(C₃F₇)₃ N], perfluorotributyl amine [(C₄ F₉)₃ N], perfluoro- tert-tributylamine, and the like,

C_(x) F_(2x-2) A₂, such as, for example, C₁₀ F₁₈ H₂, and the like,

as well as such highly fluorinated compounds as perfluoroindane,perfluoromethyl adamantane, perfluorooctyl bromide, perfluorodimethylcyclooctane, perfluoro cyclooctyl bromide, perfluoro crown ethers, andthe like.

Besides linear, branched-chain and cyclic fluorine-containing compoundsas noted above, fluorinated crown ethers (such as, for example,perfluoro 12-crown-4, perfluoro 15-crown-5, perfluoro 18-crown-6, andthe like) are also contemplated for use in the practice of the presentinvention.

In order to obtain good magnetic resonance images with high signal tonoise ratios, it is advantageous to have a high number of equivalentfluorines. As used herein, the term "equivalent fluorines" refers tothose fluorine substituents of a fluorine-containing compound whichexist in a substantially similar micro-environment (i.e., substantiallysimilar magnetic environment). Equivalent fluorines will produce oneimaging signal. A high number of equivalent fluorines will produce astrong signal, undiluted by competing signals of "non-equivalent"fluorines.

As used herein, the term "non-equivalent fluorines" refers to thosefluorine substituents of a fluorine-containing compound which exist in asubstantially dis-similar micro-environment (i.e., substantiallydissimilar magnetic environment), relative to other fluorinesubstituents on the same fluorine-containing compound. Thus, in contrastto equivalent fluorines, non-equivalent fluorines will give multiplesignals due to their different chemical shifts. Thus, while compoundswith a large number of non-equivalent fluorines are satisfactory for MRIapplications, such compounds are not ideal for maximum imaging.

Of particular interest for application to vascular imaging arefluorocarbon-containing polymeric shells having prolonged circulationtimes. Currently used angiography techniques utilize X-ray contrastmedia and are invasive procedures. The potential of ¹ H-MRI has beenrecently demonstrated for angiography applications [Edelman & Warach,New England J. of Medicine 328:785-791 (1993)]. Similarly, ¹⁹ F-MRI isuseful for angiography, with a number of advantages, such as the abilityto achieve high contrast with reference to surrounding tissue (whichdoes not contain any native fluorine). Examples of applications of suchmethodology include the diagnosis and identification of intracranialaneurysms, arteriovenous malformations, occlusions of the superior venacava, inferior vena cava, portal vein, pelvic vein, renal vein, renalmesenteric artery, peripheral mesenteric artery, and the like.

Fluorine-containing compounds entrapped in polymeric shells according tothe present invention can be used for a variety of purposes, e.g., toobtain magnetic resonance images of various organs and/or tissues, toobtain oxygen profiles in organs and/or tissues, and also to measurelocal temperature. Invention contrast agents are not limited to use inMRI applications, but can also be used for such applications asultrasonography and radiology. The other isotope of fluorine, ¹⁸ F, canbe used as a positron emission tomography (PET) contrast agent. Thus,with one fluorine-containing contrast agent, both PET and MRI diagnosiscan be accomplished. Entrapment of other imaging agents, such astechnetium and thallium compounds that are used in radiocontrast media,is also possible. Two examples of such contrast agents include Neurolyteand cardiolyte.

The use of invention compositions for oxygen detection is based upon thedramatic changes in NMR relaxation rate of ¹⁹ F in the presence of aparamagnetic species such as oxygen. Since oxygen is paramagnetic, itwill interact with the fluorine nucleus, increasing the relaxation rateof ¹⁹ F from the excited state to the normal state. By monitoring thischange in relaxation rate, it is possible to determine the oxygenconcentration in a local area (by calibrating the MRI signal to a knownconcentration of oxygen).

The novelty of this system lies, for example, in 1) the use of MRI toobtain oxygen information, 2) the use of the oxygen paramagneticinfluence on the ¹⁹ F MRI (NMR) signal, 3) the use of polymeric shellsto provide a constant and protective environment that is also permeableto oxygen, and the like.

By using fluorine-containing compounds that are solids which undergo aphase transition over physiological temperature ranges (e.g., highmolecular weight compounds, or combinations of fluorine-containingcompounds), MRI can also be used to measure local temperature.Relaxation times are much longer in solids than in liquids, thusrelaxation times will decrease dramatically as the transitiontemperature (i.e., from solid to liquid) is reached. Dramatic changesare observed in the NMR spectrum during phase transition of solid toliquid. The shape of the MRI signal for a given fluorine-containingcompound can be calibrated to a known temperature. By using a highmolecular weight fluorine-containing compound within the polymeric shell(i.e., a fluorine-containing compound having a melting point of ≧15°C.), or by using a combination of fluorine-containing compound withnon-fluorinated compound within the polymeric shell, the contents of theinterior of the polymeric shell can be selected so as to provide adesired temperature range for phase transition to occur (typically inthe range of about 22°-55° C.). The fluorocarbons within the shell willundergo a solid to liquid phase transition over the desired temperaturerange, altering substantially the observed relaxation rates, thuspermitting in vivo temperature determination. Local temperatureinformation would be especially useful, for example, in monitoringcancer patients during the hyperthermia treatment of cancer or in thedetection of cancer cells (cancer cells are cooler than normal cells).

The fluorine-containing composition employed will determine thetemperature range of the phase transition. Thus, this technique can beused over a wide temperature range, simply by changing the makeup of thefluorine-containing composition. For example, pure perfluoro-dodecane(C₁₂ F₂₆) entrapped in a polymeric shell will undergo a solid to liquidphase transition at the melting point of the fluorocarbon (75° C.).However, this transition would be sharp and only a small amount oftemperature information would be obtained. To obtain greaterinformation, the melting point of the fluorine-containing compositioncan be spread over a wider range, for example, by simply adding anothercomponent to the pure fluorine-containing composition. It is well knownin the art that a mixture will have a lower and broader melting pointrange than the corresponding pure components. Accordingly, for example,formulating perfluorododecane with a lower molecular weight fluorocarbonwill broaden the melting point range of the encapsulated composition.Similarly, a mixture of a fluorine-containing compound (e.g.,perfluorododecane) with an alkane (e.g., pentane), for example, willbroaden the melting point range of the entrapped composition.

In addition, chemically modified long chain fatty acids (e.g.,heptadecanoic acid [C₁₇ H₃₄ O₂ ], nonadecanoic acid [C₁₉ H₃₈ O₂ ], andthe like), alcohols (e.g., nonadecanol [C₁₉ H₄₀ O], Docosanol [C₂₂ H₄₆O], and the like) to which fluorines can chemically be added can also beused in the practice of the present invention. For example, adehydration coupling reaction between perfluoro-tert-butanol (t--C₄ F₉--OH; PCR CHEMICALS) with any of the above-described reactiveoxygen-containing compounds will produce a molecule that undergoes asolid to liquid phase transition and one that has nine equivalentfluorines. Similarly, a mixture of a fluorinated fatty acid andcholesterol, for example, will broaden the melting point range comparedto the pure fluorinated fatty acid, thereby allowing for localtemperature measurements to be made.

The novelty of this temperature detection system lies, for example, 1)in the use of MRI to obtain spatially resolved temperature information,2) in the use of the temperature dependence of the MRI (NMR) signal, 3)in the use of a fluorocarbon-containing composition that undergoes asolid to liquid phase transition in the desired temperature range, 4) inthe use of the polymeric shell to provide a constant and protectiveenvironment for the medium, and 5) to obtain temperature informationsimultaneously with morphology information.

According to the present invention, particles of fluorine-containingcomposition are contained within a shell having a cross-sectionaldiameter of no greater than about 10 microns (as used herein, the term"micron" refers to a unit of measure of one one-thousandth of amillimeter). A cross-sectional diameter of less than 5 microns is morepreferred, while a cross-sectional diameter of less than 1 micron ispresently the most preferred for the intravenous route ofadministration.

Contrast agents of the present invention may be introduced into the bodyspace in various ways depending on the imaging requirements. Forexample, aqueous liquid suspensions may be placed in thegastrointestinal tract by oral ingestion or suppository (e.g., to obtainimages of the stomach and gastrointestinal tract), inserted by syringeinto non-vascular spaces such as the cerebro-spinal cavity, or injectedinto the vascular system generally or into the vessels of a specificorgan such as the coronary artery. In addition, contrast agents of theinvention can also be injected into other body spaces such as theanterior and posterior eye spaces, the ear, the urinary bladder (e.g.,by way of the urethra), the peritoneal cavities, ureter, urethra, renalpelvis, joint spaces of the bone, lymphatic vessels, the subarachnoidspaces, the ventricular cavities, and the like.

The polymeric shell containing solid or liquid cores offluorine-containing composition allows for the directed delivery of highdoses of the fluorine-containing composition agent in relatively smallvolumes. This minimizes patient discomfort at receiving large volumes offluid.

In accordance with another embodiment of the present invention, there isprovided an approach to the problem of administration of substantiallywater insoluble drugs such as taxol that has not been described in theliterature. Thus, it has been discovered that delivery of such drugs canbe accomplished as an aqueous suspension of micron size particles, or anaqueous suspension containing either particles of such drug or drugdissolved in a biocompatible non-aqueous liquid. This approach wouldfacilitate the delivery of such drugs at relatively high concentrations,and thereby obviate the use of emulsifiers and their associated toxicside effects.

In accordance with yet another embodiment of the present invention, theabove-described mode of administration is facilitated by noveldrug-containing compositions wherein substantially water insoluble drugsuch as taxol is suspended in a biocompatible liquid, and wherein theresulting suspension contains particles of such drug (e.g., taxol)having a cross-sectional dimension no greater than about 10 microns. Thedesired particle size of less than about 10 microns can be achieved in avariety of ways, e.g., by grinding, spray drying, precipitation,ultrasonic irradiation, and the like.

Due to the crystal size of conventionally obtained substantially waterinsoluble drugs such as taxol, which is greater than 20 microns, solidparticles of such drugs (e.g., taxol) have not been delivered in theform of a suspension in a vehicle such as normal saline. However, thepresent invention discloses the delivery of a particulate suspension ofsubstantially water insoluble drugs (such as taxol) ground to a sizeless than about 10 microns, preferably less than about 5 microns andmost preferably less than about 1 micron, which allows intravenousdelivery in the form of a suspension without the risk of blockage in themicrocirculation of organs and tissues.

Due to the microparticular nature of the delivered drug, most of it iscleared from the circulation by organs having reticuloendothelialsystems such as the spleen, liver, and lungs. This allowspharmacologically active agents in particulate form to be targeted tosuch sites within the body.

Biocompatible liquids contemplated for use in this embodiment are thesame as those described above. In addition, parenteral nutritionalagents such as Intralipid (trade name for a commercially available fatemulsion used as a parenteral nutrition agent; available from KabiVitrum, Inc., Clayton, N.C.), Nutralipid (trade name for a commerciallyavailable fat emulsion used as a parenteral nutrition agent; availablefrom McGaw, Irvine, Calif.), Liposyn III (trade name for a commerciallyavailable fat emulsion used as a parenteral nutrition agent (containing20% soybean oil, 1.2% egg phosphatides, and 2.5% glycerin); availablefrom Abbott Laboratories, North Chicago, Ill.), and the like may be usedas the carrier of the drug particles. Alternatively, if thebiocompatible liquid contains a drug-solubilizing material such assoybean oil (e.g., as in the case of Intralipid), the drug may bepartially or completely solubilized within the carrier liquid, aidingits delivery. An example of such a case is the delivery of taxol inIntralipid as the carrier. Presently preferred biocompatible liquids foruse in this embodiment are parenteral nutrition agents, such as thosedescribed above.

In accordance with still another embodiment of the present invention,there is provided a composition for the in vivo delivery of taxolwherein taxol is dissolved in a parenteral nutrition agent.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLE 1 Preparation of Protein Shell Containing Oil

Three ml of a USP (United States Pharmacopeia) 5% human serum albuminsolution (Alpha Therapeutic Corporation) were taken in a cylindricalvessel that could be attached to a sonicating probe (Heat Systems, ModelXL2020). The albumin solution was overlayered with 6.5 ml of USP gradesoybean oil (soya oil). The tip of the sonicator probe was brought tothe interface between the two solutions and the assembly was maintainedin a cooling bath at 20° C. The system was allowed to equilibriate andthe sonicator turned on for 30 seconds. Vigorous mixing occurred and awhite milky suspension was obtained. The suspension was diluted 1:5 withnormal saline. A particle counter (Particle Data Systems, Elzone, Model280 PC) was utilized to determine size distribution and concentration ofoil-containing protein shells. The resulting protein shells weredetermined to have a maximum cross-sectional dimension of about1.35±0.73 microns, and the total concentration determined to be ˜10⁹shells/ml in the original suspension.

As a control, the above components, absent the protein, did not form astable miocroemulsion when subjected to ultrasonic irradiation. Thisresult suggests that the protein is essential for formation ofmicrospheres. This is confirmed by scanning electron micrograph andtransmission electron micrograph studies as described below.

EXAMPLE 2 Parameters Affecting Polymeric Shell Formation

Several variables such as protein concentration, temperature, sonicationtime, concentration of pharmacologically active agent, and acousticintensity were tested to optimize formation of polymeric shell. Theseparameters were determined for crosslinked bovine serum albumin shellscontaining toluene.

Polymeric shells made from solutions having protein concentrations of1%, 2.5%, 5% and 10% were counted with the particle counter to determinea change in the size and number of polymeric shells produced. The sizeof the polymeric shells was found not to vary widely with proteinconcentration, but the number of polymeric shells per ml of "milkysuspension" formed increased with the increase in concentration of theprotein up to 5%. No significant change in the number of polymericshells was found to occur above that concentration.

Initial vessel temperatures were found to be important for optimalpreparation of polymeric shells. Typically, initial vessel temperatureswere maintained between 0° C. and 45° C. The aqueous-oil interfacialtension of the oils used for formation of the polymeric shell was animportant parameter, which also varied as a function of temperature. Theconcentration of pharmacologically active agent was found not tosignificantly effect the yield of protein shells. It is relativelyunimportant if the pharmacologically active agent is incorporated in thedissolved state, or suspended in the dispersing medium.

Sonication time was an important factor determining the number ofpolymeric shells produced per ml. It was found that a sonication timegreater than three minutes produced a decrease in the overall count ofpolymeric shells, indicating possible destruction of polymeric shellsdue to excessive sonication. Sonication times less than three minuteswere found to produce adequate numbers of polymeric shells.

According to the homograph provided by the manufacturer of thesonicator, the acoustic power rating of the sonicator employed herein isapproximately 150 watts/cm². Three power settings in order of increasingpower were used, and it was found that the maximum number of polymericshells were produced at the highest power setting.

EXAMPLE 3 Preparation of Polymeric Shells Containing Dissolved Taxol

Taxol was dissolved in USP grade soybean oil at a concentration of 2mg/ml. 3 ml of a USP 5% human serum albumin solution was taken in acylindrical vessel that could be attached to a sonicating probe. Thealbumin solution was overlayered with 6.5 ml of soybean oil/taxolsolution. The tip of the sonicator probe was brought to the interfacebetween the two solutions and the assembly was maintained in equilibriumand the sonicator turned on for 30 seconds. Vigorous mixing occurred anda stable white milky suspension was obtained which containedprotein-walled polymeric shells enclosing the oil/taxol solution.

In order to obtain a higher loading of drug into the crosslinked proteinshell, a mutual solvent for the oil and the drug (in which the drug hasa considerably higher solubility) can be mixed with the oil. Providedthis solvent is relatively non-toxic (e.g., ethyl acetate), it may beinjected along with the original carrier. In other cases, it may beremoved by evaporation of the liquid under vacuum following preparationof the polymeric shells.

EXAMPLE 4 Stability of Polymeric Shells

Suspensions of polymeric shells at a known concentration were analyzedfor stability at three different temperatures (i.e., 4° C., 25° C., and38° C.). Stability was measured by the change in particle counts overtime. Crosslinked protein (albumin) shells containing soybean oil (SBO)were prepared as described above (see Example 1), diluted in saline to afinal oil concentration of 20% and stored at the above temperatures.Particle counts (Elzone) obtained for each of the samples as a functionof time are summarized in Table 2.

                  TABLE 2                                                         ______________________________________                                        Protein Shells (#/ml · 10.sup.10)                                    in saline                                                                     Day     4° C.  25° C.                                                                         38° C.                                   ______________________________________                                         0      7.9           8.9     8.1                                              1      7.4           6.9     6.8                                              7      7.3           8.3     5.0                                              9      7.8           8.1     5.8                                             17      7.8           8.3     6.1                                             23      6.9           7.8     7.4                                             27      7.2           8.8     7.1                                             ______________________________________                                    

As demonstrated by the above data, the concentration of countedparticles (i.e., polymeric shells) remains fairly constant over theduration of the experiment. The range is fairly constant and remainsbetween about 7-9·10¹⁰ /ml, indicating good polymeric shell stabilityunder a variety of temperature conditions over almost four weeks.

EXAMPLE 5 In Vivo Biodistribution--Crosslinked Protein Shells Containinga Fluorophore

To determine the uptake and biodistribution of liquid entrapped withinprotein polymeric shells after intravenous injection, a fluorescent dye(rubrene, available from Aldrich) was entrapped within a human serumalbumin (HSA) protein polymeric shell and used as a marker. Thus,rubrene was dissolved in toluene, and crosslinked albumin shellscontaining toluene/rubrene were prepared as described above byultrasonic irradiation. The resulting milky suspension was diluted fivetimes in normal saline. Two ml of the diluted suspension was theninjected into the tail vein of a rat over 10 minutes. One animal wassacrificed an hour after injection and another 24 hours after injection.

100 micron frozen sections of lung, liver, kidney, spleen, and bonemarrow were examined under a fluorescent microscope for the presence ofpolymeric shell-entrapped fluorescent dye or released dye. At one hour,the majority of the polymeric shells appeared to be intact (i.e.,appearing as brightly fluorescing particles of about 1 micron diameter),and located in the lungs and liver. At 24 hours, the dye was observed inthe liver, lungs, spleen, and bone marrow. A general staining of thetissue was also observed, indicating that the shell wall of thepolymeric shells had been digested, and the dye liberated from within.This result was consistent with expectations and demonstrates thepotential use of invention compositions for delayed or controlledrelease of an entrapped pharmaceutical agent such as taxol.

EXAMPLE 6 Toxicity of Polymeric Shells Containing Soybean Oil (SBO)

Polymeric shells containing soybean oil were prepared as described inExample 1. The resulting suspension was diluted in normal saline toproduce two different solutions, one containing 20% SBO and the othercontaining 30% SBO.

Intralipid, a commercially available TPN agent, contains 20% SBO. TheLD₅₀ for Intralipid in mice is 120 ml/kg, or about 4 ml for a 30 gmouse, when injected at 1 cc/min.

Two groups of mice (three mice in each group; each mouse weighing about30 g) were treated with invention composition containing SBO as follows.Each mouse was injected with 4 ml of the prepared suspension ofSBO-containing polymeric shells. Each member of one group received thesuspension containing 20% SBO, while each member of the other groupreceived the suspension containing 30% SBO.

All three mice in the group receiving the suspension containing 20% SBOsurvived such treatment, and showed no gross toxicity in any tissues ororgans when observed one week after SBO treatment. Only one of the threemice in the group receiving suspension containing 30% SBO died afterinjection. These results clearly demonstrate that oil contained withinpolymeric shells according to the present invention is not toxic at itsLD₅₀ dose, as compared to a commercially available SBO formulation(Intralipid). This effect can be attributed to the slow release (i.e.,controlled rate of becoming bioavailable) of the oil from within thepolymeric shell. Such slow release prevents the attainment of a lethaldose of oil, in contrast to the high oil dosages attained withcommercially available emulsions.

EXAMPLE 7 In vivo Bioavailability of Soybean Oil Released from PolymericShells

A test was performed to determine the slow or sustained release ofpolymeric shell-enclosed material following the injection of asuspension of polymeric shells into the blood stream of rats.Crosslinked protein (albumin) walled polymeric shells containing soybeanoil (SBO) were prepared by sonication as described above. The resultingsuspension of oil-containing polymeric shells was diluted in saline to afinal suspension containing 20% oil. Five ml of this suspension wasinjected into the cannulated external jugular vein of rats over a 10minute period. Blood was collected from these rats at several timepoints following the injection and the level of triglycerides (soybeanoil is predominantly triglyceride) in the blood determined by routineanalysis.

Five ml of a commercially available fat emulsion (Intralipid, an aqueousparenteral nutrition agent--containing 20% soybean oil, 1.2% egg yolkphospholipids, and 2.25% glycerin) was used as a control. The controlutilizes egg phosphatide as an emulsifier to stabilize the emulsion. Acomparison of serum levels of the trigiycerides in the two cases wouldgive a direct comparison of the bioavailability of the oil as a functionof time. In addition to the suspension of polymeric shells containing20% oil, five ml of a sample of oil-containing polymeric shells insaline at a final concentration of 30% oil was also injected. Two ratswere used in each of the three groups. The blood levels of triglyceridesin each case are tabulated in Table 3, given in units of mg/dl.

                  TABLE 3                                                         ______________________________________                                                  SERUM TRIGLYCERIDES (mg/dl)                                         GROUP       Pre    1 hr    4 hr 24 hr 48 hr                                                                              72 hr                              ______________________________________                                        Intralipid Control                                                                        11.4   941.9   382.9                                                                              15.0  8.8  23.8                               (20% SBO)                                                                     Polymeric Shells                                                                          24.8   46.7    43.8 29.3  24.2 43.4                               (20% SBO)                                                                     Polymeric Shells                                                                          33.4   56.1    134.5                                                                              83.2  34.3 33.9                               (30% SBO)                                                                     ______________________________________                                    

Blood levels before injection are shown in the column marked `Pre`Clearly, for the Intralipid control, very high triglyceride levels areseen following injection. Triglyceride levels are then seen to takeabout 24 hours to come down to preinjection levels. Thus the oil is seento be immediately available for metabolism following injection.

The suspension of oil-containing polymeric shells containing the sameamount of total oil as Intralipid (20%) show a dramatically differentavailability of detectible triglyceride in the serum. The level rises toabout twice its normal value and is maintained at this level for manyhours, indicating a slow or sustained release of triglyceride into theblood at levels fairly close to normal. The group receivingoil-containing polymeric shells having 30% oil shows a higher level oftriglycerides (concomitant with the higher administered dose) that fallsto normal within 48 hours. Once again, the blood levels of triglyceridedo not rise astronomically in this group, compared to the control groupreceiving Intralipid. This again, indicates the slow and sustainedavailability of the oil from invention composition, which has theadvantages of avoiding dangerously high blood levels of materialcontained within the polymeric shells and availability over an extendedperiod at acceptable levels. Clearly, drugs delivered within polymericshells of the present invention would achieve these same advantages.

Such a system of soybean oil-containing polymeric shells could besuspended in an aqueous solution of amino acids, essential electrolytes,vitamins, and sugars to form a total parenteral nutrition (TPN) agent.Such a TPN cannot be formulated from currently available fat emulsions(e.g., Intralipid) due to the instability of the emulsion in thepresence of electrolytes.

EXAMPLE 8 Preparation of Crosslinked Protein-walled Polymeric ShellsContaining a Solid Core of Pharmaceutically Active Agent

Another method of delivering a poorly watersoluble drug such as taxolwithin a polymeric shell is to prepare a shell of polymeric materialaround a solid drug core. Such a `protein coated` drug particle may beobtained as follows. The procedure described in Example 3 is repeatedusing an organic solvent to dissolve taxol at a relatively highconcentration. Solvents generally used are organics such as benzene,toluene, hexane, ethyl ether, and the like. Polymeric shells areproduced as described in Example 3. Five ml of the milky suspension ofpolymeric shells containing dissolved taxol are diluted to 10 ml innormal saline. This suspension is placed in a rotary evaporator at roomtemperature and the volatile organic removed by vacuum. After about 2hours in the rotary evaporator, these polymeric shells are examinedunder a microscope to reveal opaque cores, indicating removal ofsubstantially all organic solvent, and the presence of solid taxolwithin a shell of protein.

Alternatively, the polymeric shells with cores of organicsolvent-containing dissolved drug are freeze-dried to obtain a drycrumbly powder that can be resuspended in saline (or other suitableliquid) at the time of use. In case of other drugs that may not be inthe solid phase at room temperature, a liquid core polymeric shell isobtained. This method allows for the preparation of a crosslinkedprotein-walled shell containing undiluted drug within it. Particle sizeanalysis shows these polymeric shells to be smaller than thosecontaining oil. Although the presently preferred protein for use in theformation of the polymeric shell is albumin, other proteins such asα-2-macroglobulin, a known opsonin, could be used to enhance uptake ofthe polymeric shells by macrophage-like cells. Alternatively, aPEG-sulfhydryl (described below) could be added during formation of thepolymeric shell to produce a polymeric shell with increased circulationtime in vivo.

EXAMPLE 9 In vivo Circulation and Release Kinetics of Polymeric Shells

Solid core polymeric shells containing taxol were prepared as describedabove (see, for example, Example 3) and suspended in normal saline. Theconcentration of taxol in the suspension was measured by HPLC asfollows. First, the taxol within the polymeric shell was liberated bythe addition of 0.1 M mercaptoethanol (resulting in exchange of proteindisulfide crosslinkages, and breakdown of the crosslinking of thepolymeric shell), then the liberated taxol was extracted from thesuspension with acetonitrile. The resulting mixture was centrifuged andthe supernatant freeze-dried. The lyophilate was dissolved in methanoland injected onto an HPLC to determine the concentration of taxol in thesuspension. The taxol concentration was found to be about 1.6 mg/ml.

Rats were injected with 2 ml of this suspension through a jugularcatheter. The animal was sacrificed at two hours, and the amount oftaxol present in the liver determined by HPLC. This requiredhomogenization of the liver, followed by extraction with acetonitrileand lyophilization of the supernatant following centrifugation. Thelyophilate was dissolved in methanol and injected onto an HPLC.Approximately 15% of the administered dose of taxol was recovered fromthe liver at two hours, indicating a significant dosage to the liver.This result is consistent with the known function of thereticuloendothelial system of the liver in clearing small particles fromthe blood.

EXAMPLE 10 Preparation of Crosslinked PEG-walled Polymeric Shells

As an alternative to the use of thiol (sulfhydryl) containing proteinsin the formation of, or as an additive to polymeric shells of theinvention, a thiol-containing PEG was prepared. PEG is known to benontoxic, noninflammatory, nonadhesive to cells, and in generalbiologically inert. It has been bound to proteins to reduce theirantigenicity and to liposome forming lipids to increase theircirculation time in vivo. Thus incorporation of PEG into an essentiallyprotein shell would be expected to increase circulation time as well asstability of the polymeric shell. By varying the concentration ofPEG-thiol added to the 5% albumin solution, it was possible to obtainpolymeric shells with varying stabilities in vivo. PEG-thiol wasprepared by techniques available in the literature (such as thetechnique of Harris and Herati, as described in Polymer Preprints Vol.32:154-155 (1991)).

PEG-thiol of molecular weight 2000 g/mol was dissolved at aconcentration of 1% (0.1 g added to 10 ml) in a 5% albumin solution.This protein/PEG solution was overlayered with oil as described inExample 1 and sonicated to produce oil-containing polymeric shells withwalls comprising crosslinked protein and PEG. These polymeric shellswere tested for stability as described in Example 4.

Other synthetic water-soluble polymers that may be modified with thiolgroups and utilized in lieu of PEG include, for example, polyvinylalcohol, polyhydroxyethyl methacrylate, polyacrylic acid,polyethyloxazoline, polyacrylamide, polyvinyl pyrrolidinone,polysaccharides (such as chitosan, alginates, hyaluronic acid, dextrans,starch, pectin, and the like), and the like.

For example, fluorocarbon-containing protein shells having prolongedcirculation times in vivo were found to have particular benefit forimaging the vascular system. These shells remained within thecirculation for extended periods, relative to shells not containing PEGin the shell walls. This allowed, for example, visulation of cardiaccirculation, and provided a non-invasive means of evaluating thecoronary circulation, instead of using conventional invasive techniquessuch as angiography.

EXAMPLE 11 Targeting of Immunosuppressive Agent to Transplanted Organsusing Intravenous Delivery of Polymeric Shells Containing Such Agents

Immunosuppressive agents are extensively used following organtransplantation for the prevention of rejection episodes. In particular,cyclosporine, a potent immunosuppressive agent, prolongs the survival ofallogeneic transplants involving skin, heart, kidney, pancreas, bonemarrow, small intestine, and lung in animals. Cyclosporine has beendemonstrated to suppress some humoral immunity and to a greater extent,cell mediated reactions such as allograft rejection, delayedhypersensitivity, experimental allergic encephalomyelitis, Freund'sadjuvant arthritis, and graft versus host disease in many animal speciesfor a variety of organs. Successful kidney, liver and heart allogeneictransplants have been performed in humans using cyclosporine.

Cyclosporine is currently delivered in oral form either as capsulescontaining a solution of cyclosporine in alcohol, and oils such as cornoil, polyoxyethylated glycerides and the like, or as a solution in oliveoil, polyoxyethylated glycerides, and the like. It is also administeredby intravenous injection, in which case it is dissolved in a solution ofethanol (approximately 30%) and Cremaphor (polyoxyethylated castor oil)which must be diluted 1:20 to 1:100 in normal saline or 5% dextroseprior to injection. Compared to an intravenous (i.v.) infusion, theabsolute bioavailibility of the oral solution is approximately 30%(Sandoz Pharmaceutical Corporation, Publication SDI-Z10 (A4), 1990). Ingeneral, the i.v. delivery of cyclosporine suffers from similar problemsas the currently practiced i.v. delivery of taxol, i.e., anaphylacticand allergic reactions believed to be due to the Cremaphor, the deliveryvehicle employed for the i.v. formulation. In addition, the intravenousdelivery of drug (e.g., cyclosporike) encapsulated as described hereavoids dangerous peak blood levels immediately following administrationof drug. For example, a comparison of currently available formulationsfor cyclosporine with the above-described encapsulated form ofcyclosporine showed a five-fold decrease in peak blood levels ofcyclosporine immediately following injection.

In order to avoid problems associated with the Cremaphor, cyclosporinecontained within polymeric shells as described above may be delivered byi.v. injection. It may be dissolved in a biocompatible oil or a numberof other solvents following which it may be dispersed into polymericshells by sonication as described above. In addition, an importantadvantage to delivering cyclosporine (or other immunosuppressive agent)in polymeric shells has the advantage of local targeting due to uptakeof the injected material by the RES system in the liver. This may, tosome extent, avoid systemic toxicity and reduce effective dosages due tolocal targeting. The effectiveness of delivery and targeting to theliver of taxol contained within polymeric shells following intravenousinjection is demonstrated in Example 9. A similar result would beexpected for the delivery of cyclosporine (or other putativeimmunosuppressive agent) in accordance with the present invention.

EXAMPLE 12 Antibody Targeting of Polymeric Shells

The nature of the polymeric shells of the invention allows for theattachment of monoclonal or polyclonal antibodies to the polymericshell, or the incorporation of antibodies into the polymeric shell.Antibodies can be incorporated into the polymeric shell as the polymericmicrocapsule shell is being formed, or antibodies can be attached to thepolymeric microcapsule shell after preparation thereof. Standard proteinimmobilization techniques can be used for this purpose. For example,with protein microcapsules prepared from a protein such as albumin, alarge number of amino groups on the albumin lysine residues areavailable for attachment of suitably modified antibodies. As an example,antitumor agents can be delivered to a tumor by incorporating antibodiesagainst the tumor into the polymeric shell as it is being formed, orantibodies against the tumor can be attached to the polymericmicrocapsule shell after preparation thereof. As another example, geneproducts can be delivered to specific cells (e.g., hepatocytes orcertain stem cells in the bone marrow) by incorporating antibodiesagainst receptors on the target cells into the polymeric shell as it isbeing formed, or antibodies against receptors on the target cells can beattached to the polymeric microcapsule shell after preparation thereof.In addition, monoclonal antibodies against nuclear receptors can be usedto target the encapsulated product to the nucleus of certain cell types.

EXAMPLE 13 Polymeric Shells as Carriers for Polynucleotide Constructs,Enzymes and Vaccines

As gene therapy becomes more widely accepted as a viable therapeuticoption (at the present time, over 40 human gene transfer proposals havebeen approved by NIH and/or FDA review boards), one of the barriers toovercome in implementing this therapeutic approach is the reluctance touse viral vectors for the incorporation of genetic material into thegenome of a human cell. Viruses are inherently toxic. Thus, the risksentailed in the use of viral vectors in gene therapy, especially for thetreatment of non-lethal, non-genetic diseases, are unacceptable.Unfortunately, plasmids transferred without the use of a viral vectorare usually not incorporated into the genome of the target cell. Inaddition, as with conventional drugs, such plasmids have a finite halflife in the body. Thus, a general limitation to the implementation ofgene therapy (as well as antisense therapy, which is a reverse form ofgene therapy, where a nucleic acid or oligonucleotide is introduced toinhibit gene expression) has been the inability to effectively delivernucleic acids or oligonucleotides which are too large to permeate thecell membrane.

The encapsulation of DNA, RNA, plasmids, oligonucleotides, enzymes, andthe like, into protein microcapsule shells as described herein canfacilitate their targeted delivery to the liver, lung, spleen, lymph andbone marrow. Thus, in accordance with the present invention, suchbiologics can be delivered to intracellular locations without theattendant risk associated with the use of viral vectors. This type offormulation facilitates the non-specific uptake or endocytosis of thepolymeric shells directly from the blood stream to the cells of the RES,into muscle cells by intramuscular injection, or by direct injectioninto tumors. In addition, monoclonal antibodies against nuclearreceptors can be used to target the encapsulated product to the nucleusof certain cell types.

Diseases that can be targeted by such constructs include diabetes,hepatitis, hemophilia, cystic fibrosis, multiple sclerosis, cancers ingeneral, flu, AIDS, and the like. For example, the gene for insulin-likegrowth factor (IGF-1) can be encapsulated into protein microcapsuleshells for delivery for the treatment of diabetic peripheral neuropathyand cachexia. Genes encoding Factor IX and Factor VIII (useful for thetreatment of hemophilia) can be targeted to the liver by encapsulationinto protein microcapsule shells of the present invention. Similarly,the gene for the low density lipoprotein (LDL) receptor can be targetedto the liver for treatment of atherosclerosis by encapsulation intoprotein microcapsule shells of the present invention.

Other genes useful in the practice of the present invention are geneswhich re-stimulate the body's immune response against cancer cells. Forexample, antigens such as HLA-B7, encoded by DNA contained in a plasmid,can be incorporated into a protein microcapsule shell of the presentinvention for injection directly into a tumor (such as a skin cancer).Once in the tumor, the antigen will recruit to the tumor specific cellswhich elevate the level of cytokines (e.g., IL-2) that render the tumora target for immune system attack.

As another example, plasmids containing portions of the adeno-associatedvirus genome are contemplated for encapsulation into proteinmicrocapsule shells of the present invention. In addition, proteinmicrocapsule shells of the present invention can be used to delivertherapeutic genes to CD8+ T cells, for adoptive immunotherapy against avariety of tumors and infectious diseases.

Protein microcapsule shells of the present invention can also be used asa delivery system to fight infectious diseases via the targeted deliveryof an antisense nucleotide, for example, against the hepatitis B virus.An example of such an antisense oligonucleotide is a 21-merphosphorothioate against the polyadenylation signal of the hepatitis Bvirus.

Protein microcapsule shells of the present invention can also be usedfor the delivery of the cystic fibrosis transmembrane regulator (CFTR)gene. Humans lacking this gene develop cystic fibrosis, which can betreated by nebulizing protein microcapsule shells of the presentinvention containing the CFTR gene, and inhaling directly into thelungs.

Enzymes can also be delivered using the protein microcapsule shells ofthe present invention. For example, the enzyme, DNAse, can beencapsulated and delivered to the lung. Similarly, ribozymes can beencapsulated and targeted to virus envelop proteins or virus infectedcells by attaching suitable antibodies to the exterior of the polymericshell. Vaccines can also be encapsulated into polymeric microcapsules ofthe present invention and used for subcutaneous, intramuscular orintravenous delivery.

EXAMPLE 14 Preparation of Insoluble Hemoglobin Constructs (IHC) for useas a Red Blood Cell Substitute

A 20 ml glass reaction cell, titanium horn and collar were washed withalcohol and sterile saline prior to synthesis as was all equipment used.In a typical reaction, 3.5 ml of 5% w/v hemoglobin (human or bovine) wasadded to a reaction cell which was attached to the ultrasonic horn (HeatSystems XL2020, 20 KHz, 400 W maximum power). The horn and cell werethen submerged in a temperature control bath set to 55° C. Reactions runat 55° C. appeared to be optimum, however product can be synthesizedover a wide range of temperatures (0° to 80° C.). The pH was 6.8.Temperature control is critical to high yields of material, and theoptimum temperature depends on the specific experimental configuration.The ultrasonic source turned on at a power setting of 7. Using themanufacturer's nomograph suggested a power output of approximately 150W/cm². The reaction is complete in about 30 seconds. Yields at shorterand longer reaction times appear to be less. For bovine hemoglobin, the2.5% w/v solution was passed through a Sephadex G-25 gel permeationcolumn to remove any anions such as phosphates. In a typical synthesisof human hemoglobin IHC, the ultrasonic horn was positioned at theair-water interface. The homogeneous suspension produced containsproteinaceous red blood cells. The aqueous suspension may then be storedin a sterile container at 4° C.

A typical reaction yields a solution that contains approximately 3×10⁸IHC shells per ml with an average shell diameter of 3 microns with astandard deviation of 1 micron. This synthetic procedure yields highconcentrations of micron-sized biomaterial with narrow sizedistributions.

After the synthesis, the IHC remain as a suspension in the nativeprotein solution. To separate the IHC from the unreacted protein,several methods were used: filtration, centrifugation and dialysis. Thefirst method included filtering the mixture through an Anotop syringefilter with 0.2 μm diameter pore size (Whatman, Inc.). The filter waswashed with several volumes of water until the filtrate contained verylittle or no protein (as determined by UV-Visible spectroscopy). The IHCwere "backwashed" out of the filter and resuspended in an equivalentvolume of saline. The second purification procedure involved the use ofa Centricon centrifuge filter with a molecular-weight cut-off of 100kilodaltons (kD). The centrifuge filter is a centrifuge tube separatedby a filtration membrane in the middle. Centrifugation of the IHCsolution at 1000 G for 5 minutes allowed most of the unreactedhemoglobin (64.5 kD) to pass through the membrane. Finally, dialysiswith a large molecular weight (300 kD) membrane was also used to purifythe IHC. However, this method required approximately 2 days of dialysis.The preferred method for the purification of the IHC is with theCentricon centrifugation.

EXAMPLE 15 Preparation of an Insoluble Hemoglobin/Albumin Construct(IHAC) as a Red Blood Cell Substitute

A 20 ml glass reaction cell, titanium horn and collar were washed withalcohol and sterile saline prior to synthesis as was all equipment used.In a typical reaction, 3.5 ml of a 5% w/v hemoglobin and albumin (humanor bovine; hemoglobin/albumin ratio varied from 0.5 to 2) was added to areaction cell which was attached to the ultrasonic horn (Heat SystemsXL2020, 20 KHz, 400 W maximum power). The horn and cell were thensubmerged in a temperature control bath set to 55° C. Reactions run at55° C. appeared to be optimum, however product can be synthesized over awide range of temperatures (0° to 80° C.). The pH was 6.8. Temperaturecontrol is critical to high yields of material, and the optimumtemperature depends on the specific experimental configuration. Theultrasonic source turned on at a power setting of 7. Using themanufacturer's nomograph suggested a power output of approximately 150W/cm². The reaction is complete in about 30 seconds. Yields at shorterand longer reaction times appear to be less. The homogeneous suspensionproduced contains the proteinaceous red blood cell substitute. Theaqueous suspension was filtered, washed, resuspended in sterile bufferedsaline and stored in a sterile container at 4° C.

Again as described above, typical reaction yields a solution thatcontains roughly 10⁸ shells per ml with an average shell diameter of 3microns with a standard deviation of 1 micron. This synthetic procedureyields high concentrations of micron-sized biomaterial with narrow sizedistributions.

Alternately a flow-through system that allows the continuous processingof the IHC can be utilized. Such a system consists of peristaltic pumpsthat continously pump streams of hemoglobin and optionally abiocompatible oil or fluorocarbon into a reaction vessel with asonicator probe. A suitable residence time is maintained in the vesseland the IHC recovered by overflow from the vessel into a recovery tank.The unreacted hemoglobin solution is recycled into the reaction vessel.

EXAMPLE 16 Preparation of Insoluble Hemoglobin Constructs ContainingEncapsulated Fluorocarbons

A 20 ml glass reaction cell, titanium horn and collar were washed withalcohol and sterile saline prior to synthesis as was all equipment used.In a typical reaction, 3.5 ml of a 5% w/v hemoglobin (human or bovine)was added to a reaction cell which was attached to the ultrasonic horn(Heat Systems XL2020, 20 KHz, 400 W maximum power). A fluorocarbon,perfluorodecalin 3.5 ml, was added to the reaction vessel. The horn andcell were then submerged in a temperature control bath set to 20° C. ThepH of the aqueous phase was 6.8. The ultrasonic source turned on at apower setting of 7. Using the manufacturer's nomograph suggested a poweroutput of approximately 150 W/cm². The reaction is complete in about 30seconds. The homogeneous suspension produced contains the microcapsulesor microspheres of crosslinked insoluble hemoglobin shells withencapsulated perfluorodecalin in the interior. The milky suspension isfiltered, washed, resuspended in sterile buffered saline as above andstored in a sterile container at 4° C.

Again as described above, typical reaction yields a solution thatcontains roughly 10⁸ shells per ml with an average shell diameter of 3microns with a standard deviation of 1 micron. This synthetic procedureyields high concentrations of micron-sized biomaterial with narrow sizedistributions.

EXAMPLE 17 Preparation of Insoluble Albumin Constructs ContainingEncapsulated Fluorocarbons

A 20 ml glass reaction cell, titanium horn and collar were washed withalcohol and sterile saline prior to synthesis as was all equipment used.In a typical reaction, 3.5 ml of a 5% w/v albumin (human or bovine) wasadded to a reaction cell which was attached to the ultrasonic horn (HeatSystems XL2020, 20 KHz, 400 W maximum power). A fluorocarbon,perfluorodecalin (or perfluorotripropyl amine) 3.5 ml, was added to thereaction vessel. The horn and cell were then submerged in a temperaturecontrol bath set to 20° C. The pH of the aqueous phase was 6.8. Theultrasonic source turned on at a power setting of 7. Using themanufacturer's nomograph suggested a power output of approximately 150W/cm². The reaction is complete in about 30 seconds. The homogeneoussuspension produced contains the microcapsules or microspheres ofcrosslinked insoluble Albumin shells with encapsulated perfluorodecalin(or perfluorotripropyl amine) in the interior. The milky suspension isfiltered, washed, resuspended in sterile buffered saline as above andstored in a sterile container at 4° C.

Again as described above, typical reaction yields a solution thatcontains roughly 10⁸ shells per ml with an average shell diameter of 3microns with a standard deviation of 1 micron. This synthetic procedureyields high concentrations of micron-sized biomaterial with narrow sizedistributions.

EXAMPLE 18 Insoluble Hemoglobin Constructs further Modified withAllosteric Modifiers such as Pyridoxal 5'-Phosphate (PLP)

In order to obtain hemoglobin constructs with variable affinities tooxygen (i.e., variable P₅₀), the IHC were further reacted with PLP, aknown allosteric modulator. A suspension of IHC (obtained as in Examplein tris buffer was deoxygenated at 10 C under nitrogen. 10 ml of thedeoxygenated IHC suspension was taken in each of six separate reactionvessels. Different molar ratios of PLP/Hb were added to each of thevessels. They were 0.1/3.0, 0.75/3.0, 1.5/3.0, 3.0/3.0, 4.2/3.0,6.0/3.0. After 30 minutes, a tenfold excess of sodium borohydride isadded an allowed to reduce the Schiff's base for another 30 minutes. Thesuspension is then filtered by centrifugation, backwashed 3 times withbuffered saline, resuspended in buffered saline and stored at 4° C. Thismodification targets the amino terminal groups of the b-globin chain indeoxyhemoglobin. In this respect the modification closely mimics theaction of 2,3-DPG (which binds at lysine EF₆ (82)b) in stabilizing thedeoxy confirmation.

The six different degrees of modification will result in IHC withincreasing P₅₀ (decreasing oxygen affinities) with increasing degree ofPLP substitution.

EXAMPLE 19 Insoluble Constructs with Crosslinked Shells of Hemoglobinand Polyethylene Glycol

Polyethylene glycol (PEG) is known to be nontoxic, noninflammatory,nonadhesive to cells, and in general biologically inert. Proteins thatare attached with PEG have been found to be less antigenic. Withliposomes, circulation was found increased upon binding/incorporation ofPEG. Thus incorporation of PEG into the RBC will be expected to increasecirculation time. By varying the concentration of PEG-thiol added to theprotein (e.g., hemoglobin), it was possible to prepare PEG-hemoglobinRBC that had varying stabilities. The PEG-thiol was prepared bytechniques in the literature (such as Harris and Heart Polymer Preprints32:154 (1991).

PEG-thiol of molecular weight 2000 g/mol was dissolved at aconcentration of 1% (0.1 g added to 10 ml) in a 5% hemoglobin solution.The protein-peg solution was sonicated to form the proteinaceous redblood cell substitute as described in Example 14.

EXAMPLE 20 Insoluble Hemoglobin Constructs with Polyethylene GlycolCovalently Attached to the Shell Exterior

The IHC were prepared as described in Example 14. Polyethylene glycol ofMW10,000 (PEG 10 k) was reacted with 1,1'-Carbonyl diimidazole CDIaccording to techniques available in the literature (Beauchamp et al.Analytical Biochemistry 131: 25-33, 1983). The IHC were suspended in 50mM borate buffer pH 8.0 and PEG-CDI (2 fold molar excess relative tototal hemoglobin lysines) was added and the reaction mixture stirred atroom temperature for 6 hours. The resulting PEG-IHC were then separatedby filtration, washed in saline and resuspended in sterile bufferedsaline.

EXAMPLE 21 Parameters Affecting Formation of Insoluble HemoglobinConstructs

Several variables such as protein concentration, temperature, sonicationtime, acoustic intensity, pH were tested to optimize formation of theIHC.

These materials were prepared from 1%, 2.5%, 5%, and 10% hemoglobinsolutions. They were also prepared from mixed protein solution such ashemoglobin and human serum albumin with concentrations again rangingfrom 1 to 10%. The size and concentrations was determined with aparticle counter. The size was found not to significantly vary withstarting protein concentration. The number prepared increased withincrease starting protein concentration up to about 5%. No significantchange in the number was found to occur above that concentration.

Initial vessel temperature were found to be important for optimalpreparation of the IHC. Typically the initial reaction temperatures weremaintained between 0° and 80° C. The optimal starting temperature wasroughly 70° C.

Sonication time was also important factor determining the number of IHCproduced per ml. It was found that a sonication time of roughly 30seconds was good for synthesizing a high concentration of the IHC. Longor shorter sonication times produced less but still a adequate number ofIHC.

According to the nomograph provided by the manufacture of the sonicator,the acoustic power rating of the sonicator used in these experiments isapproximately 150 watts/cm². Other power setting were also found toproduce a large number of IHC.

EXAMPLE 22 Insoluble Hemoglobin Constructs as Drug Carriers ofOil-Soluble Drugs

The cytotoxic effects of several antineoplastic drugs are greatlyenhanced in the presence of oxygen. It is therefore desirable to delivera drug to a tumor site while increasing oxygen concentration at thatsite. The hemoglobin microspheres of the present invention allow forthat capability. Example 16 above describes the encapsulation of afluorocarbon liquid in a shell of insoluble hemoglobin. Cytotoxic drugssuch as cyclophosphamide, BCNU, Melphalan, taxol, camptothecin,adriamycin, etoposide, and the like, can be dissolved in thefluorocarbon or other suitable oil such as soybean oil and encapsulatedinto the hemoglobin construct.

Taxol was dissolved in soybean oil (SBO) at a concentration of 5 mg/ml.3.5 ml of a 5% hemoglobin solution was taken in a to a reaction vesseland 3.5 ml of the SBO/taxol was added to the vessel. The two phasemixture was sonicated as described in Example 16 to obtain crosslinkedinsoluble hemoglobin shells containing SBO/Taxol.

EXAMPLE 23 Polymeric Shells as Drug Carriers of Water-Soluble Drugs

Several water-soluble drugs are candidates for encapsulation intopolymeric shells. As an example methotrexate was dissolved in water at aconcentration of 5 mg/ml. One ml of this aqueous solution was emulsifiedwith 4 ml of soybean oil using Pluronic-65 (block copolymer ofpolyethylene oxide and polypropylene oxide) to form a stablewater-in-oil (W/O) microemulsion. 3.5 ml of a 5% hemoglobin solution wasoverlayered with 3.5 ml of this W/O microemulsion and sonicated for 30seconds to obtain insoluble hemoglobin constructs containing anencapsulated microemulsion with methotrexate.

EXAMPLE 24 Polymeric Shells as Protein Carriers

Several proteins are candidates for encapsulation into polymeric shells,e.g., hemoglobin, albumin, and the like. For example, as a method ofincreasing the hemoglobin loading of the IHC, hemoglobin could beencapsulated into the IHC instead of the water soluble drug in Example23. hemoglobin was dissolved in water at a concentration of 10%. One mlof this aqueous solution was emulsified with 4 ml of soybean oil usingPluronic-65 (block copolymer of polyethylene oxide and polypropyleneoxide) to form a stable water-in-oil (W/O) microemulsion. 3.5 ml of a 5%Hemoglobin solution was overlayered with 3.5 ml of this W/Omicroemulsion containing hemoglobin. The two phase mixture was sonicatedfor 30 seconds to obtain insoluble hemoglobin constructs containing anencapsulated microemulsion that also contained hemoglobin. This methodserved to increase the total amount of hemoglobin per microsphere of theIHC and therefore increased the oxygen carrying capacity for boundoxygen.

EXAMPLE 25 In Vivo Administration of Albumin/FluorocarbonConstructs-Magnetic Resonance Imaging (¹⁹ F-MRI) to DetectBiodistribution

Albumin constructs containing perfluorononane were prepared as inExample 17. The final suspension was made up to contain 20% by volume ofthe fluorocarbon in sterile saline. Two ml of this suspension wasinjected via the tail vein injection into a ketamine anesthetizedSprague Dawley rat. The in vivo distribution of the fluorocarbon wasmonitored by ¹⁹ F-MRI on a Bruker 500 MHz NMR instrument. The rat wasplaced into a 10 cm ¹⁹ F coil and images obtained using a T₁ weightedsequence with TR=1 second, TE=20 milliseconds, and a data matrix of256×128.

At 1 hour after administration most of the FC was found to accumulate inthe liver, lungs, and spleen. Some of the FC could also be detected inthe bone marrow. Hemoglobin constructs would be expected to behave in anidentical fashion in terms of tissue localization and accumulation.These observations had important implications for the treatment of liverand lung tumors and possibly the treatment of neoplastic cells in thebone marrow with high doses of oxygen in conjunction with the localdelivery of a cytotoxic drug or as an adjuvant to radiation therapy.

EXAMPLE 26 In Vivo Administration of Drug Carrying Constructs

Insoluble hemoglobin constructs containing encapsulated Taxol (in SBO)were prepared as in Example 22. The final suspension was made up tocontain 20% by volume of the SBO in sterile saline. 2 ml of thissuspension was injected via the tail vein injection into a ketamineanesthetized Sprague Dawley rat.

The rat was sacrificed 2 hours after the injection and the liverrecovered. The liver was homogenized with a small volume of saline andextracted with ethyl acetate. The extract was lyophilized, dissolved inmethanol and injected into an HPLC column. Approximately 15% of theinitial dose of unmetabolized taxol was recovered from the liver. Thisdetermined the feasibility of targeting antineoplastic drugs to theliver in conjunction with the delivery of oxygen to these sites.

EXAMPLE 27 Acute Blood Replacement Model for Insoluble Hemoglobin BloodSubstitute

Anesthetized Sprague-Dawley rats (350-400g) are catheterized through theexternal jugular vein. Approximately 70% of their blood volume isremoved over a period of 10 minutes. The rats are maintained in thisstate for 10 additional minutes following which they are reinfused withan iso-oncotic suspension of oxygenated IHC with a P₅₀ of 28 mm Hg. Themean arterial pressure, heart rate and breathing rate are continouslymonitored. The survival of these rats is followed over time.

EXAMPLE 28 Insoluble Hemoglobin Constructs for Reversal of TissueIschemia

The ability of the IHC to preferentially deliver oxygen to an ischemicsite is exploited. IHC with `high affinity`, i.e., P₅₀ <28 mm Hg areuseful for this purpose since they will release oxygen only at siteswhere oxygen gradients are larger than normally encountered in thecirculation, that is to say, at an ischemic site. An IHC with P₅₀ of 20mm Hg is utilized for this purpose.

A bilateral carotid occlusion model in a rat is used as a model of`Stroke` or cerebral ischemia. Both carotid arteries are occluded bytemporary ligature in a ketamine anesthetized Sprague-Dawley rat. In thecontrol rat, the ligature is removed after 15 minutes and normal bloodflow is resumed. In the experimental rat, 1 ml of a high affinity IHCsuspension in saline is infused directly into each carotid arteryfollowing external oxygenation of the IHC suspension in an oxygenationdevice. 24 hours after the treatment, the rats are sacrificed, theirbrains retrieved, fixed, sectioned and stained with nitro bluetetrazolium (NBT) or trypan blue to determine the degree of cell death.A lower degree of cell death, as determined by tryptan blue staining, isexpected in the experimental rat receiving invention IHC.

EXAMPLE 29 Evaluation of In Vivo Circulation Half-Life of InsolubleHemoglobin Constructs

Anesthetized Sprague-Dawley rats (350-400 g) are catheterized throughthe external jugular vein. A bolus injection of an iso-oncoticsuspension of IHC equivalent to 20% of the animals' blood volume isgiven through the catheter. Blood is withdrawn at sampling times rangingfrom 0.25 to 92 hours. Blood samples are centrifuged and plasma observedfor signs of hemolysis or presence of soluble hemoglobin. Since the`microbubbles` of the IHC have a gaseous interior (and are therefore oflower density than water), they rise to the surface of the plasmafollowing centrifugation. The microbubbles are skimmed off, resuspendedin saline and counted in a particle counter. The half-lives of IHC incirculation is then determined. Compared to prior art hemoglobin-basedblood substitutes, it is expected that invention IHC will demonstrateenhanced circulation half life.

EXAMPLE 30 IHC for Organ Preservation-Preservation of the Rat Heart

The heart is surgically removed from an anesthetized Sprague-Dawley ratand artificially respirated with room air. The heart is immersed incrystalloid medium (`Cardioplegia medium`--CM) having the samecomposition as IHC (or IHC/FC, or Albumin/FC) preservation medium butwithout the hemoglobin component. The heart is perfused with the CM forseveral minutes, cooling it to 11° C. The heart is then preserved with140 ml of IHC preservation medium for 12 hours at 12° C. The IHC mediumis continously perfused through the heart at a low pressure (18 mm Hg)and continously equilibriated with 95% O2/5% CO₂. After 12 hours ofpreservation, the contractile, pump, and energetic functioning of theheart is tested using an isolated working rat heart apparatus.

EXAMPLE 31 Utility of IHC Media in Cardioplegia for Open Heart Surgery

Cardiopulmonary bypass is instituted and oxygenated `cardioplegiamedium` containing IHC (or IHC/FC, or Albumin/FC) as an oxygen carrier,at 4° C., is delivered as a bolus of 500 to 100 ml into the aortic rootafter appropriate aortic cross-clamping and venting. Additional doses ofthe cold medium are delivered to the left and right coronary ostia, andin the case of bypass surgeries, the medium is also delivered into theends of the grafts prior to final anastomoses. The medium is deliveredevery 15 to 20 min in quantities sufficient to maintain a coolmyocardial temperature. After completing the procedure, the aortic clampis removed and rewarming of the heart started.

EXAMPLE 32 Utility of IHC Media in Angioplasty or Atherectomy

The IHC (or IHC/FC, or Albumin/FC) medium is administered duringinterventional procedures undertaken to restore flow to obstructed orunderperfused regions of an organ. Examples of such procedures areangioplasty and atherectomy. Regional ischemia can be mitigated duringballoon inflation of the percutaneous transluminal coronary angioplastyprocedure by delivering oxygenated IHC medium at a rate of about 60ml/min through the central lumen of the dilating balloon catheter. Themedium is administered at body temperature and contains, for example,physiologically compatible Ringer's electrolytes and substrates. A doseof oxygen equilibriated IHC medium is infused during each ballooninflation period. A similar procedure is used during the period ofballoon inflation in atherectomy procedures which are used to physicallyremove obstructions in vessels by knife or laser. Infusion of the mediumdirectly into the obstructed vessel during enzymatic thrombolyticprocedures could be done to provide oxygenation distal to theobstruction as it is lysed. Currently Fluosol-DA is used during someangioplasty procedures; the IHC (or IHC/FC, or Albumin/FC) medium of thepresent invention would replace Fluosol-DA.

EXAMPLE 33 Synthesis of Dodecafluorononane (C₉ F₂₀) Entrapped within aPolymeric Shell

A 20 ml glass reaction cell, titanium horn and collar were washed withalcohol and sterile saline prior to synthesis as was all equipment used.In a typical reaction, 3.5 ml of sterile 5% w/v USP (United StatesPharmacopaeia) human serum albumin (Alpha Therapeutics Corporation) wasadded to a reaction cell and the cell attached to the ultrasonic horn(Heat Systems XL2020, 20 KHz, 400 W maximum power). The horn and cellwere then submerged in a temperature control bath set to 22° C.Reactions run at 22° C. appeared to be optimum, however product can besynthesized over a wide range of temperatures (0° up to about 40° C.).Temperature control is critical to high yields of material, and theoptimum temperature depends on the specific experimental configuration.

Six milliliters of dodecafluorononane (C₉ F₂₀) was next added, and theultrasonic source turned on at a power setting of 7. The amount offluorocarbon added can be varied from less than one ml up to about 13 mlwith good yield of protein polymeric shells. The reaction is complete inabout 30 seconds. Yields at shorter and longer reaction times appear tobe less. The homogeneous suspension produced contains the entrappeddodecafluorononane in protein polymeric shells and is approximately 60%perfluorononane by volume. The aqueous suspension may then be stored ina sterile container at 4° C.

A typical reaction yields a solution that contains approximately 1×10⁹shells per mL with an average shell diameter of 2 microns with astandard deviation of 1 micron. This synthetic procedure is seen toyield high concentrations of micron-sized biomaterial with narrow sizedistributions.

EXAMPLE 34 Synthesis of Perfluorotributyl amine (C₁₂ F₂₇ N) orPerfluorotripropyl amine (C₉ F₂₁ N) Entrapped within Polymeric Shells

The 5% w/v USP human serum albumin (3.5 ml) and fluoroamine (6 ml) wereadded to a glass reaction cell and irradiated with high intensityultrasound. The reaction conditions were a power setting of 7, a bathtemperature of 22° C. and a reaction time of approximately 30 seconds.Once again high concentration of both perfluorotripropyl amine [(C₃ F₇)₃N] and perfluorotributyl amine [(C₄ F₉)₃ N] entrapped in a proteinpolymeric shell are synthesized (1×10⁹ shells/mL) with an averagediameter of 2 microns.

EXAMPLE 35 Synthesis of Perfluorodecalin (C₁₀ F₁₈) Entrapped within aPolymeric Shell

The 5% w/v USP human serum albumin (3.5 ml) and perfluorodecalin (C₁₀F₁₈ ; 6 ml) were added to a glass reaction cell and irradiated with highintensity ultrasound. The reaction conditions were a power setting of 7,a bath temperature of 22° C. and a reaction time of approximately 30seconds. High concentration with narrow size distributions ofperfluorodecalin contained within a protein polymeric shell weresynthesized. Furthermore, since perfluordecalin andperfluorotripropylamine are the major constituents of the FDA approvedfluorocarbon, Fluosol DA, the medicinal use of these compounds inmedical imaging should be readily accepted by regulatory authorities.

EXAMPLE 36 Synthesis of Perfluoro 15-crown-5 (C₁₀ F₂₀ O₅) Entrappedwithin a Polymeric Shell

The 5% w/v USP human serum albumin (3.5 ml) and the fluorocrown ether(C₁₀ F₂₀ O₅ ; 6 ml) were added to a glass reaction cell and irradiatedwith high intensity ultrasound. The reaction conditions were a powersetting of 7, a bath temperature of 22° C. and a reaction time ofapproximately 30 seconds. As before, high concentrations of fluorocrownether contained in a protein polymeric shell with narrow sizedistributions are synthesized. In fact this experimental procedure tosynthesize fluorocarbon filled polymeric shells was typical for all ofthe fluorocarbons investigated.

EXAMPLE 37 Synthesis of Perfluoro-t-butylbutene (C₁₀ F₁₈ H₂) Entrappedwithin a Polymeric Shell

The 5% w/v USP human serum albumin (3.5 ml) and C₁₀ F₁₈ H₂ (6 ml) can beadded to a glass reaction cell and irradiated with high intensityultrasound. Reaction conditions comprising a power setting of 7, a bathtemperature of 22° C. and a reaction time of approximately 30 secondswould typically be employed. By this procedure, protein polymeric shellhaving a high concentration of fluoro-t-butylbutane entrapped thereincould be synthesized.

EXAMPLE 38 Toxicity of Fluorocarbons Contained within Polymeric Shells

Five rats were injected through a catherized jugular vein with 5 ml of a20% v/v fluorocarbon suspension (perfluorononane contained in an HSAprotein polymeric shell) over 10 minutes. Fluorocarbons in general arenontoxic due the strong fluorine-carbon bonds; indeed, fluorocarbonshave been successfully used as FDA approved artificial blood substitutes(Fluosol DA). The rats were harvested at specific times and autopsied.Besides observing the general health of the rat, the liver, spleen,lungs and kidneys were carefully examined. Rats examined at 0.5, 2, 8and 24 hours were all healthy with no inflamed tissues or organs. Thefifth rat is still alive and healthy after 90 days. For comparison, thisdose of FDA approved soybean oil in a rat is the LD₅₀ amount, furthersuggesting that fluorocarbons are nontoxic and safe.

EXAMPLE 39 ¹⁹ F Nuclear Magnetic Resonance Spectroscopy of a NeatFluorocarbon and a Fluorocarbon Entrapped within a Polymeric Shell

NMR spectra of the fluorocarbons contained within a protein polymericshell and neat fluorocarbons were obtained on a Bruker 500 MHz NMRinstrument. The instrument was tuned for ¹⁹ F at its resonance frequencyof 470.56 MHz. A deuterium solvent was used for locking and all spectrawere externally referenced to Freon (CCl₃ F) at 0 ppm. Perfluorononaneand CDCl₃ were placed in a 5 mm NMR tube. The spectrum of pureperfluorononane was obtained with two sets of sharp peaks, one at -87ppm, and the second set of peaks at -127, -128, and -133 ppm.

A suspension of perfluorononane entrapped within HSA protein polymericshells was resuspended in D₂ O and a similar NMR spectrum was obtained.Strong signals were obtained from the 20% v/v fluorocarbon suspensionwith peaks or resonances at -81, -121, -122 and -126 ppm. The entrapmentof the fluorocarbon in the polymeric shell during ultrasonic irradiationresulted in no chemical or structural changes of the perfluorononane.For example, with C₉ F₂₀ two separate resonance were observed: onecorresponding to the CF₃ at approximately -80 ppm and the second set ofresonances at approximately -125 ppm, corresponding to the CF₂ group.

EXAMPLE 40 ¹⁹ F Nuclear Magnetic Resonance Spectroscopy of Fluorocarbonsto Measure Local Temperature

Variable temperature NMR spectra of fluorocarbons were obtained on aBruker 500 MHz NMR instrument. The instrument was tuned for ¹⁹ F at itsresonance frequency of 470.56 MHz. A deuterium solvent (d6-dimethylsulfoxide [d₆ -DMSO]) was used for locking and all spectra wereexternally referenced to freon (CCl₃ F) at 0 ppm. Perfluorododecane,which has a melting point of 77° C., and d₆ -DMSO were placed in a 5 mmNMR tube at room temperature. Fluorine spectra were collected atdifferent temperatures and the linewidths were measured. Linewidth dataat -81 ppm, as a function of temperature, are shown below:

    ______________________________________                                        Linewidth @ -81 ppm (Hz)                                                                        Temperature (°C.)                                    ______________________________________                                        51.1              102                                                         57.0              82                                                          64.65             60                                                          ______________________________________                                    

The broad spectrum at lower temperatures starts to sharpen as thetemperature increases, resulting from the perfluorododecane undergoingits solid to liquid phase transition. The change is sharp and suddenwith temperature, as expected for a pure material.

In order to broaden and lower the melting temperature, pentane was added(approximately 2% v/v) to the perfluorododecane. As was seen above, thebroad spectra at lower temperatures sharpened as the Rerfluorododecanegoes through its solid to liquid phase transition. Linewidth data as afunction of temperature for the perfluorododecane/pentane mixture areshown below:

    ______________________________________                                        Linewidth (Hz)                                                                -82 ppm      -123.3 ppm                                                                              Temperature (°C.)                               ______________________________________                                        21.26        87.17     77                                                     165.89       280.50    67                                                     216.6        341.2     57                                                     290.77       436.15    47                                                     578.27       451.33    37                                                     577.62       525.11    27                                                     ______________________________________                                    

The resulting perfluorododecane/pentane mixture has a lower meltingpoint that is broadened as expected. With this system, temperaturemeasurements can be made in the range from 27° to 77° C. Thus, given alinewidth, it is possible to determine the local temperature.

An example of use of this technique to determine localized temperaturesin vivo involves the injection of protein shells containing fluorocarbonmixtures (e.g., such as described above) with broad melting transitionshaving temperature-linewidth correlations (which can be empiricallyobtained). Such a formulation will localize within the liver or spleenand, in addition to serving as a ¹⁹ F MRI contrast agent, maysimultaneously be utilized to determine locally variant temperatureswithin the organ (allowing the elucidation of the pathology ofsignificant abnormalities within the tissues).

EXAMPLE 41 ¹⁹ F Magnetic Resonance Imaging of Phantoms

Two types of entrapped fluorocarbons contained in polymeric shells wereused in this phantom study. Perfluorononane and perfluorotributyl aminecontained within HSA protein polymeric shells were synthesized asdescribed in Examples 33 and 34. The synthesized suspension that was 60%fluorocarbon per volume was diluted with saline and 2 milliliters placedin polystyrene tubes. The polystyrene tubes were than placed in acommercially available Siemens 2T MRI instrument (10 cm ¹⁹ F coil)operating at 1.5 tesla. ¹⁹ F magnetic resonance images of the tubes weretaken over a 5 minute period with an echo time (TE) of 10 millisecondsand a time of repetition (TR) of 300 seconds (256×256 matrix).

    ______________________________________                                        Perflurononane Contained in Polymeric Shells                                  Dilution  [conc], M       Image Clarity                                       ______________________________________                                        1         1.8             excellent                                           1/2       0.9             excellent                                           1/4       0.45            good                                                 1/10     0.18            good                                                 1/50     0.09            good                                                 1/100    0.02            marginal                                            ______________________________________                                    

Good MR phantom images were observed even at low concentrations ofperfluorononane entrapped within polymeric shells. Very similar data wasobserved with polymeric shells that contained perfuorotributyl amine.Only at high dilution (1/100; 0.02 M) was the image of poor quality andresolution.

EXAMPLE 42 ¹⁹ F Magnetic Resonance Imaging of Liver and Spleen In Vitro

300 gram rats were injected with 2 ml of 20% v/v perfluorononanecontained within an HSA protein polymeric shell suspension. At 2 hoursand at 5 days, a rat was sacrificed and the liver, spleen, kidneys, andlungs were removed. The entire liver, for example, was then placed in a4 tesla MRI instrument operating with a 10 cm ¹⁹ F coil. ¹⁹ F magneticresonance images of the liver, spleen and kidney were obtained using aT₁ weighted sequence with a TR=1 second, a TE=20 milliseconds and a datamatrix of 256×128 (i.e., 128 phase encoding steps, 16 signal averages).

¹⁹ F MRI images of the liver showed regions of varying intensity whichcorrelated to varying degrees of liver uptake of the polymeric shells.For example, a dark region corresponding to the portal vein was observedwhere one would not expect the presence of theperfluorononane-containing polymeric shells since most of the shells areconcentrated intracellularly within the RES of the liver.

The average image intensity of the liver scan at two hours afterinjection was approximately 20-30% higher than that of a scan recorded 5days after injection, indicating partial dissipation of theperfluorononane, possibly through breakdown of the polymeric shells.Overall, excellent quality images showing liver morphology wereobtained, demonstrating the potential of this technique in the diagnosisand localization of abnormal pathology within the liver.

EXAMPLE 43 In Vivo ¹⁹ F Magnetic Resonance Imaging of Liver and Spleen

A 150 gram rat was injected with 2 ml of a 20% v/v perfluorononane (C₉F₂₀) contained within HSA polymeric shells over 10 minutes. The entirerat was then placed in a 4 tesla MRI instrument operating with a 10 cm¹⁹ F coil. The rat was anaesthetized with ketamine before collectingimages. ¹⁹ F magnetic resonance images of the entire rat, as well asindividual organs such as the liver, spleen and kidney, were obtainedusing a T₁ weighted sequence with a TR=1 second, a TE=20 milliseconds,and a data matrix of 256×128 (i.e., 128 phase encoding steps, 16 signalaverages).

Rats were imaged 15 minutes, 2 hours, and 24 hours after injection ofthe perfluorononane-containing HSA protein polymeric shells. Overall,excellent quality images showing liver and spleen morphology wereobtained, demonstrating the potential of this technique in the diagnosisand localization of abnormal pathology within the liver RES containingorgans.

EXAMPLE 44 Determination of Local Temperature using In Vivo ¹⁹ FMagnetic Resonance Imaging

A 300 gram rat is injected with 5 ml of a 20% v/v perfluorododecane/2%pentane (or perfluorononadecanoic acid and 1% cholesterol) containedwithin HSA polymeric shells over 10 minutes. The rat is then placed in a15 cm coil (a Siemens 1.5 tesla MRI magnet). ATE of 10 milliseconds andTR of 300 seconds is used to collect the images (256×256 matrix). Therat is anaesthetised with ketamine before collecting data. The liver andspleen are imaged over a 15 minute period, by taking a 5 millimeterslice thickness. Data are collected at room temperature and atapproximately 37° C., by wrapping the subdued rat in a heating pad.

EXAMPLE 45 In Vivo Oxygen Determination Using ¹⁹ F Magnetic ResonanceImaging

A 300 gram rat is injected with 5 ml of 20% v/v perfluorononanecontained within HSA polymeric shells over 10 minutes. The rat is nextplaced in a 15 cm coil (a Siemens 1.5 tesla MRI magnet). ATE of 70milliseconds and TR of 3 seconds is used to collect the images (256×256matrix). The rat is placed in a restraining harness before collectingdata. The rat is first put in an oxygen chamber to increase oxygenmetabolism, and the linewidth and image are collected. The rat is nextinjected with ketamine, to reduce the consumption of oxygen, and againthe linewidth and image are collected. The linewidth and the intensityof the image are observed to change, corresponding to the amount ofdissolved oxygen in the rat. The largest linewidth is observed at higheroxygen concentrations. The liver and spleen are imaged over 15 minutestaking a 5 millimeter slice thickness. Two data sets are collected, oneat room temperature and another at 37° C., by wrapping the anaesthetizedrat in a heating pad.

EXAMPLE 46 Preparation of Taxol Particles

Crystals of taxol (Sigma Chemical) were ground in a ball mill untilparticles of solid taxol were obtained having a size less than 10microns. Size of particles were determined by suspending the particlesin isotonic saline and counting with the aid of a particle counter(Elzone, Particle Data). Grinding was continued until 100% of theparticles had a size less than 5 microns. The preferred particle sizefor intravenous delivery is less than 5 microns and most preferably lessthan 1 micron.

Alternatively, particles of taxol were obtained by sonicating asuspension of taxol in water until all particles were below 10 microns.

Taxol particles less than 10 microns can also be obtained byprecipitating taxol from a solution of taxol in ethanol by adding wateruntil a cloudy suspension is obtained. Optionally, the solution of taxolcan be sonicated during the water addition, until a cloudy suspension isobtained. The resulting suspension is then filtered and dried to obtainpure taxol particles in the desired size range.

Fine particles of taxol were prepared by spray drying a solution oftaxol in a volatile organic such as ethanol. The solution was passedthrough an ultrasonic nozzle that formed droplets of ethanol containingtaxol. As the ethanol evaporated in the spray drier, fine particles oftaxol were obtained. Particle size can be varied by changing theconcentration of taxol in ethanol, adjusting the flow rate of liquidthrough the nozzle and power of sonication.

EXAMPLE 47 Synthesis of Paramagnetic Cations Bound to Polyanions

Synthesis of Gd-alginates can be carried out, for example, by dispersingthe alginate in a solution of GdCl₃. For example, small sphericalparticles of Gd-alginate suitable for intravascular injection may besynthesized by ultrasonic irradiation of a solution containing Gd ions(e.g., GdCl₃) and adding small quantities of Na-alginate solution. Thealginate is dispersed into the solution of Gd ions by the ultrasonicirradiation, and crosslinked by the multivalent Gd ions, producingmicron sized particles of Gd-alginate. Besides ultrasonic irradiation,low or high speed mixing can also be used.

Alternatively, a solution of Na-alginate is overlaid or layered on animmiscible organic solvent or oil (e.g., soybean oil, sunflower oil,toluene, methylene chloride, chloroform, and the like). The liquids aresubjected to ultrasonic irradiation whereby the alginate-containingaqueous phase is dispersed into the organic phase, then a solution ofmultivalent ions (e.g., GdCl₃, MnCl₃, FeCl₃, and the like) is added. TheNa-alginate is thereby crosslinked, producing tiny spherical particlesof Gd-alginate which are suitable for use as an MRI contrast agentfollowing intravascular injection. Essentially any synthetic techniqueusing alginates and multivalent cations can be used to form spheres,fibers, plates, blocks, and the like.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

That which is claimed is:
 1. A method for the preparation of articlesfor in vivo delivery of nutriceuticals, said method comprisingsubjecting aqueous medium containing biocompatible material capable ofbeing crosslinked by disulfide bonds and a nutriceutical to highintensity ultrasound conditions for a time sufficient to promotecrosslinking of said biocompatible material by disulfide bonds;whereinsaid nutriceutical is substantially contained within a polymeric shell,and wherein the largest cross-sectional dimension of said shell is nogreater than about 10 microns.
 2. The method according to claim 1,wherein said biocompatible material is a naturally occurring polymer, asynthetic polymer, or a combination thereof,wherein said polymer, priorto crosslinking, has covalently attached thereto sulfhydryl groups ordisulfide linkages.
 3. The method according to claim 1, wherein saidnutriceutical is selected from amino acids, sugars, proteins,carbohydrates, fat-soluble vitamins, fat, or combinations of any two ormore thereof.
 4. The method according to claim 1, wherein saidnutriceutical within said shell is dissolved or suspended in abiocompatible dispersing agent.
 5. A method for the delivery of anutriceutical to a subject in need thereof, said method comprisingadministering to said subject the articles prepared by the method ofclaim 1 by oral, intravenous, subcutaneous, intraperitoneal,intrathecal, intramuscular, intracranial, inhalational, topical,transdermal, suppository (rectal), or pessary (vaginal) routes ofadministration.
 6. A method for the preparation of articles for in vivodelivery of nutriceuticals, said method comprising subjecting aqueousmedium containing a nutriceutical capable of being crosslinked bydisulfide bonds to high intensity ultrasound conditions for a timesufficient to promote crosslinking of said biocompatible material bydisulfide bonds to form a polymeric shell;wherein the largestcross-sectional dimension of said shell is no greater than about 10microns.
 7. The method according to claim 6, wherein said nutriceuticalis a naturally occurring polymer, a synthetic polymer, or a combinationthereof,wherein said polymer, prior to crosslinking, has covalentlyattached thereto sulfhydryl groups or disulfide linkages.
 8. The methodaccording to claim 6, wherein said nutriceutical is selected from aminoacids, sugars, proteins, carbohydrates, fat-soluble vitamins, fat, orcombinations of any two or more thereof.
 9. A method for the delivery ofa nutriceutical to a subject in need thereof, said method comprisingadministering to said subject the articles prepared by the method ofclaim 1 by oral, intravenous, subcutaneous, intraperitoneal,intrathecal, intramuscular, intracranial, inhalational, topical,transdermal, suppository (rectal), or pessary (vaginal) routes ofadministration.